Altered polypeptides with increased half-life

ABSTRACT

Polypeptides that are cleared from the kidney and do not contain in their original form a Fc region of an IgG are altered so as to comprise a salvage receptor binding epitope of an Fc region of an IgG and thereby have increased circulatory half-life.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to polypeptides that are mutated to contain asalvage receptor binding epitope. More particularly, this inventionrelates to polypeptides that are cleared through the kidney having anepitope from the Fc region of an IgG molecule, resulting in longercirculatory half-life.

2. Description of Related Literature

It was proposed in 1964 that a specific receptor exists in rapidequilibrium with the intravascular space that protects IgG moleculesfrom degradation. Brambell et al., Nature, 203: 1352-1355 (1964). Seealso Brambell, The Lancet, 1087-1093 (1965). The kidney has been shownto be the major site of catabolism of immunoglobulin fragments,according for approximately 90% of their endogenous catabolism. Wochneret al., J. Exp. Med., 126: 207 (1967). The existence of a receptorimplies that the Ig molecule has specific sequences, or conformationaldeterminants, that must bind to such a receptor. Since the Fc region ofIgG produced by proteolysis has the same in vivo half-life as the intactIgG molecule and Fab fragments are rapidly degraded (Spiegelberg andWiegle, J. Exp. Med., 121: 323-338 [1965]; Waldmann and Ghetie,“Catabolism of Immunoglobulins,” Progress in Immunol., 1: 1187-1191[Academic Press, New York: 1971]; Spiegelberg, in Advances inImmunology, Vol. 19, F. J. Dixon and H. G. Kinkel, ads. [Academic Press,NY: 1974], pp. 259-294; and reviewed by Zuckier et al., Semin. Nucl.Med., 19: 166-186 [1989]), it was believed that the relevant sequencesof mouse IgG_(2b) were in the CH2 or CH3 domain and that deletion of oneor the other domain would give rise to rapid degradation. In fact, theCH2 domain fragment of human IgG produced by trypsin digestion of the Fcfragment persisted in the circulation of rabbits for as long as the Fcfragment or IgG molecule; in contrast, the CH3 domain (pFc′) fragment ofhuman IgG also produced by trypsin digestion of the Fc fragment wasrapidly eliminated, indicating that the catabolic site of IgG is locatedin the CH2 domain. Ellerson et al., J. Immunol., 116: 510 (1976);Yasmeen et al., J. Immunol., 116: 518 (1976). Other studies have shownthat sequences in the CH3 domain are important in determining thedifferent intravascular half-lives of IgG_(2b)T and IgG_(2a)h antibodiesin the mouse. Pollock et al., Eur. J. Immunol., 20: 2021-2027 (1990).

The catabolic rates of IgG variants that do not bind the high-affinityFc receptor FcRI or C1q are indistinguishable from the rate of clearanceof the parent wild-type antibody, indicating that the catabolic site isdistinct from the sites involved in FcRI or C1q binding. Wawrzynczak etal., Molec. Immunol., 29: 221 (1992). Also, removal of carbohydrateresidues from the IgG molecule or Fc fragment has either a minor role inor no effect on the in vivo half-life, and the extent of this effectdepends on the isotype of the IgG molecule. Nose and Wigzell, Proc.Natl. Acad. Sci. USA, 80: 6632 (1983); Tao and Morrison, J. Immunol.,143: 2595 (1989); Wawrzynczak et al., Mol. Immunol., 29: 213 (1992).

Staphylococcal protein A-IgG complexes were found to clear more rapidlyfrom the serum than uncomplexed IgG molecules. Dima et al., Eur. J.Immunol., 13: 605 (1983). To determine if residues near the Fc-SpAinterface are involved in IgG clearance, Kim et al., Eur. J. Immunol.,24: 542-548 (1994) performed site-directed mutagenesis to change aminoacid residues of a recombinant Fc-hinge fragment derived from the murineimmunoglobulin G1 molecule and determine the effects of these mutationson the pharmacokinetics of the Fc-hinge fragment. The authors showedthat the site of the IgG1 molecule that controls the catabolic rate (the“catabolic site”) is located at the CH2-CH3 domain interface andoverlaps with the Staphylococcal protein A binding site. See also WO93/22332 published Nov. 11, 1993. The concentration catabolismphenomenon is also studied in Zuckier et al., Cancer, 73: 794-799(1994). IgG catabolism is also discussed by Masson, J. Autoimmunity, 6:683-689 (1993).

WO 94/04689 discloses a protein with a cytotoxic domain, aligand-binding domain and a peptide linking these two domains comprisingan IgG constant region domain having the property of increasing thehalf-life of the protein in mammalian serum.

A stereo drawing of a human Fc fragment and its complex with fragment Bof Protein A from Staphylococcus aureus is provided by Deisenhofer,Biochemistry, 20: 2364 (1981).

It has been shown that clearance is greatly reduced when the effectivemolecular size exceeds 70 kDa, the glomerular filtration cutoff size.Knauf et al., “Relationship of Effective Molecular Size to SystemicClearance in Rats of Recombinant Interleukin-2 Chemically Modified withWater-soluble Polymers,” J. Biochem., 263: 15064-15070 (1988).

SUMMARY OF THE INVENTION

Accordingly, in one embodiment the invention provides a polypeptidevariant of a polypeptide of interest which polypeptide of interest iscleared from the kidney and does not contain a Fc region of an IgG,which variant comprises a salvage receptor binding epitope of an Fcregion of an IgG, and which variant has a longer in vivo half-life thanthe polypeptide of interest.

In another aspect, the invention provides nucleic acid encoding thepolypeptide variant, a replicable vector comprising the nucleic acid, ahost cell comprising the nucleic acid, and a method for producing apolypeptide variant comprising culturing the host cells in culturemedium and recovering the polypeptide variant from the host cellculture. The nucleic acid molecule may be labeled or unlabeled with adetectable moiety.

In a further aspect, the invention supplies a polypeptide that is not anFc, which polypeptide comprises one or more of the sequences (5′ to 3′):HQNLSDGK (SEQ ID NO: 1), HQNISDGK (SEQ ID NO: 2), or VISSHLGQ (SEQ IDNO: 31), and which polypeptide also comprises the sequence: PKNSSMISNTP(SEQ ID NO: 3).

In a still further aspect, the invention provides a method for preparinga polypeptide variant comprising altering a polypeptide of interest thatis cleared from the kidney and does not contain an Fc region of an IgGso that it comprises a salvage receptor binding epitope of an Fc regionof an IgG and has an increased in vivo half-life.

In a still additional embodiment, the invention supplies a method forpreparing a polypeptide variant having an increased in vivo half-lifecomprising:

(1) identifying the sequence and conformation of a salvage receptorbinding epitope on an Fc region of an IgG molecule;

(2) altering the sequence of a polypeptide of interest that is clearedfrom the kidney and does not contain an Fc region to include thesequence and conformation of the identified binding epitope;

(3) testing the altered polypeptide of step (2) for longer in vivohalf-life than that of the polypeptide of interest; and

(4) if the polypeptide does not have a longer in vivo half-life, furtheraltering the sequence of the polypeptide of interest to include thesequence and conformation of the identified binding epitope and testingfor longer in vivo half-life until longer in vivo half-life is obtained.

In a still further aspect, the invention provides a method for treatingan LFA-1-mediated disorder comprising administering to a mammal,preferably a patient, in need of such treatment an effective amount ofthe variant set forth above wherein the polypeptide is a Fab, a (Fab′)₂,a diabody, a Fv fragment, a single-chain Fv fragment, or a receptor andacts as an LFA-1 antagonist. More preferably, this variant is a Fab or(Fab′)₂ of anti-LFA-1 [such as an anti-CD18 Fab or (Fab′)₂], withincreased serum half-life as set forth herein.

In another embodiment, the invention provides a method for detectingCD11a or CD18 in vitro or in vivo comprising contacting the anti-CD11aor CD18 antibody fragment variant herein with a sample, especially aserum sample, suspected of containing the CD11a or CD18 and detecting ifbinding has occurred.

The Fc region is to be located (transplanted) to a region of thepolypeptide of interest that will not alter its conformation so that itloses biological activity and is to be located so that it will notinterfere with the polypeptide's ability to bind with a ligand orantigen to maintain biological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the serum pharmacokinetics of five Fab or (Fab′)₂constructs in mice after single intravenous doses of 2 mg/kg. In FIG.1A, the Fab v1B variant is designated by solid squares, the Fab controlis indicated by solid diamonds, the Fab v2 variant is indicated by solidtriangles, the Fab v1 variant is indicated by solid circles, and thedouble-disulfide F(ab′)₂ is indicated by open circles. In FIG. 1B, theFab control is designated as solid triangles, the variant Fab v2 isdesignated by open circles; the variant Fab v1 is designated by opensquares; the variant Fab v1B is designated by solid circles; and thedouble-disulfide F(ab′)₂ is designated by solid squares. The moleculesare more fully described in the tables herein.

FIG. 2 depicts an alignment of the relevant portions of the consensusamino acid sequences of the human IgG1 CH1 domain (SEQ ID NO: 4), thehuman IgG2 CH1 domain (SEQ ID NO: 5), the human IgG3 CH1 domain (SEQ IDNO: 6), the human IgG4 CH1 domain (SEQ ID NO: 7), the human kappa CLdomain (SEQ ID NO: 8), and the human lambda CL domain (SEQ ID NO: 9), inalignment with the Fab v1b variant derived from anti-CD18 antibody (SEQID NO: 10), which is described in Example 1. In this figure, amino acidresidues and/or positions of interest and of most importance to theinvention within the sequence of Fab v1b (i.e., SEQ ID NOS: 3 and 1) aredesignated by underlining and asterisks, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

As used herein, “polypeptide of interest” refers to a polypeptide thathas a biological activity, is cleared from the kidney, and does notcontain a Fc region of an IgG. An “Fc region of an IgG” refers to the Fcportion of an immunoglobulin of the isotype IgG, as is well known tothose skilled in the art of antibody technology. Examples of suchpolypeptides are peptides and proteins, whether from eukaryotic sourcessuch as, e.g., yeast, avians, plants, insects, or mammals, or frombacterial sources such as, e.g., E. coli. The polypeptide of interestmay be isolated from natural sources or made synthetically orrecombinantly. In a preferred embodiment, the polypeptide of interestcontains an Ig domain or Ig-like domain, e.g., an antigen-bindingdomain.

Clearance of polypeptides of interest from the kidney depends at leastin part on the molecular weight of the polypeptide. Polypeptides of toolarge a molecular weight will not clear the kidneys of a mammal. Oneexample of a test to determine whether the polypeptide of interest (orvariant) clears the kidney is a clinical study wherein the polypeptideof interest or variant is labeled with a detectable marker andadministered to the same type of mammal that will be treated, using atreatment regimen the same as would be used in the actual treatment.Thereafter, a clinical sample of the urine of the mammal is taken andanalyzed to determine if the label is detected therein. If the label isdetected, the polypeptide of interest or variant has cleared thekidneys.

As a general rule, polypeptides clearing the kidney have a molecularweight in the range of about 5,000 daltons, although molecules withsomewhat higher or lower molecular weights may also meet the criteria ofthis invention if they can pass the renal clearance test noted above.

The polypeptide of interest is biologically active if it has an in vivoeffector or antigenic function or activity that is directly orindirectly caused or performed by the polypeptide (whether in its nativeor denatured conformation) or a fragment thereof. Effector functionsinclude receptor binding and any carrier binding activity, agonism orantagonism of the polypeptide of interest, especially transduction of aproliferative signal including replication, DNA regulatory function,modulation of the biological activity of various growth factors,receptor activation, deactivation, up- or down-regulation, cell growthor differentiation, and the like. Biological activity includespossession of an epitope or antigenic site that is capable ofcross-reacting with antibodies raised against the polypeptide ofinterest or mammalian equivalents thereof.

Examples of mammalian polypeptides of interest include molecules suchas, e.g., renin, a growth hormone, including human growth hormone;bovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; α1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; thrombopoietin; folliclestimulating hormone; calcitonin; luteinizing hormone; glucagon; clottingfactors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnaturietic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; a serum albumin such as humanserum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; DNase; inhibin; activin; vascularendothelial growth factor (VEGF); receptors for hormones or growthfactors; integrin; protein A or D; rheumatoid factors; a neurotrophicfactor such as brain-derived neurotrophic factor (BDNF); neurotrophin-3,-4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor suchas NGF-β; cardiotrophins (cardiac hypertrophy factor) such ascardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF);fibroblast growth factor such as aFGF and bFGF; epidermal growth factor(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins; CD proteins such as CD-3,CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;an anti-HER-2 antibody without a native Fc region of an IgG; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressing;regulatory proteins; antibodies without a native Fc region of an IgG;and fragments of any of the above-listed polypeptides.

The preferred polypeptides of interest are mammalian polypeptides.Examples of such mammalian polypeptides include antibody fragments suchas Fv, Fab, (Fab′)₂, and an anti-HER-2 fragment without the IgGFcdomain, t-PA, gp120, DNase, IGF-I, IGF-II, brain IGF-1, growth hormone,relaxin chains, growth hormone releasing factor, insulin chains orpro-insulin, urokinase, immunotoxins, neurotrophins, and antigens. Morepreferably, the polypeptide is a Fab, a (Fab′)₂, a diabody, a Fvfragment, a single-chain Fv fragment, or a receptor. Even morepreferably, the polypeptide is an anti-IgE, anti-HER2, or anti-CD18 Fabor (Fab′)₂, and most preferably is human or humanized.

As used herein, “polypeptide variant” refers to an amino acid sequencevariant of the polypeptide of interest, including variants with one ormore amino acid substitutions, insertions, and/or deletions. Suchvariants are biologically active as defined above and necessarily haveless than 100% sequence identity with the polypepide of interest. In apreferred embodiment, the biologically active polypeptide variant has anamino acid sequence sharing at least about 70% amino acid sequenceidentity with the polypeptide of interest, preferably at least about75%, more preferably at least about 80%, still more preferably at leastabout 85%, even more preferably at least about 90%, and most preferablyat least about 95%.

“In vivo half life” means the half-life of the polypeptide of interestor polypeptide variant circulating in the blood of a given mammal.

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, andIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule. As an example, FIG. 2 shows representative epitopes inunderlining and the important residues in asterisks. The IgG1, IgG2, andIgG4 isotypes are preferred for determining the salvage receptor bindingepitope.

“Polymerase chain reaction” or “PCR” refers to a procedure or techniquein which minute amounts of a specific piece of nucleic acid, RNA and/orDNA, are amplified as described in U.S. Pat. No. 4,683,195 issued 28Jul. 1987. Generally, sequence information from the ends of the regionof interest or beyond needs to be available, such that oligonucleotideprimers can be designed; these primers will be identical or similar insequence to opposite strands of the template to be amplified. The 5′terminal nucleotides of the two primers may coincide with the ends ofthe amplified material. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA, and cDNAtranscribed from total cellular RNA, bacteriophage or plasmid sequences,etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol.,51: 263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).As used herein, PCR is considered to be one, but not the only, exampleof a nucleic acid polymerase reaction method for amplifying a nucleicacid test sample comprising the use of a known nucleic acid as a primerand a nucleic acid polymerase to amplify or generate a specific piece ofnucleic acid.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains (Clothiaet al., J. Mol. Biol., 186: 651-663 [1985]; Novotny and Haber, Proc.Natl. Acad. Sci. USA, 82: 4592-4596 [1985]).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., supra). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and binding site. This region consists of a dimer ofone heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

“Single-chain Fv” or “sFv”antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun, A. in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “antibody” is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies), antibody compositions with polyepitopic specificity,bispecific antibodies, diabodies, and single-chain molecules, as well asantibody fragments (e.g., Fab, F(ab′)₂, and Fv), so long as they exhibitthe desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler and Milstein, Nature, 256: 495 (1975), or may bemade by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567[Cabilly et al.]). The “monoclonal antibodies” may also be isolated fromphage antibody libraries using the techniques described in Clackson etal., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (Cabilly et al., supra;Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 [1984]).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details see: Joneset al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).The humanized antibody includes a Primatized™ antibody wherein theantigen-binding region of the antibody is derived from an antibodyproduced by immunizing macaque monkeys with the antigen of interest.

“Non-immunogenic in a human” means that upon contacting the polypeptideof interest or polypeptide variant in a pharmaceutically acceptablecarrier and in a therapeutically effective amount with the appropriatetissue of a human, no state of sensitivity or resistance to thepolypeptide of interest or variant is demonstrable upon the secondadministration of the polypeptide of interest or variant after anappropriate latent period (e.g., 8 to 14 days).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) on thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Holliger et al.,Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

The term “LFA-1-mediated disorders” refers to pathological states causedby cell adherence interactions involving the LFA-1 receptor onlymphocytes. Examples of such disorders include T cell inflammatoryresponses such as inflammatory skin diseases including psoriasis;responses associated with inflammatory bowel disease (such as Crohn'sdisease and ulcerative colitis); adult respiratory distress syndrome;dermatitis; meningitis; encephalitis; uveitic; allergic conditions suchas eczema and asthma and other conditions involving infiltration of Tcells and chronic inflammatory responses; skin hypersensitivityreactions (including poison ivy and poison oak); atherosclerosis;leukocyte adhesion deficiency; autoimmune diseases such as rheumatoidarthritis, systemic lupus erythematosus (SLE), diabetes mellitus,multiple sclerosis, Reynaud's syndrome, autoimmune thyroiditis,experimental autoimmune encephalomyelitis, Sjorgen's syndrome, juvenileonset diabetes, and immune responses associated with delayedhypersensitivity mediated by cytokines and T-lymphocytes typically foundin tuberculosis, sarcoidosis, polymyositis, granulomatosis andvasculitis; pernicious anemia; diseases involving leukocyte diapedesis;CNS inflammatory disorder, multiple organ injury syndrome secondary tosepticaemia or trauma; autoimmune haemolytic anemia; myethemia gravis;antigen-antibody complex mediated diseases; all types oftransplantations, Including graft vs. host or host vs. graft disease;hemorrhagic shock; pulmonary oxygen toxicity; pulmonary fibrosis; woundrepair; B-cell lymphomas; etc.

In particular, the preferred indications for antibodies to CD11a orCD11b are psoriasis, transplant rejection, asthma, wound repair, andpulmonary fibrosis; the preferred indications for antibodies to CD18 arehemorrhagic shock, meningitis; encephalitis; multiple sclerosis; asthma;and pulmonary oxygen toxicity; and the preferred indication forantibodies to CD20 is B-cell lymphoma.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein which the disorder is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, sheep, pigs, cows, etc.Preferably, the mammal herein is human.

The term “LFA-1 antagonist” generally refers to an antibody directedagainst either CD11a or CD18 or both, but also includes soluble forms ofICAM-1 (e.g., the ICAM-1 extracellular domain), antibodies to ICAM-1,and fragments thereof, or other molecules capable of inhibiting theinteraction of LFA-1 and ICAM-1.

The term “anti-LFA-1 antibody” or “anti-LFA-1 MAb” refers to an antibodydirected against either CD11a or CD18 or both. The anti-CD11a antibodiesinclude, e.g., MHM24 (Hildreth et al., Eur. J. Immunol., 13: 202-208[1983]), R3.1 (IgG1; Rothlein, Boehringer Ingelheim Pharmaceuticals,Inc., Ridgefield, Conn.), 25-3 (or 25.3; an IgG1 available fromImmunotech, France; see Olive et al., in Feldmann, ed., Human T cellClones. A new Approach to Immune Regulation, Clifton, N.J., Humana,[1986] p. 173), KBA (IgG2a; Nishimura et al., Cell. Immunol., 107: 32[1987]; Nishimura et al., Cell. Immunol., 94: 122 [1985]), M7/15 (IgG2b;Springer et al., Immunol. Rev., 68: 171 [1982]), IOT16 (Vermot Desrocheset al., Scand. J. Immunol., 33: 277-286 [1991]), SPVL7 (Vermot Desrocheset al., supra), and M17 (IgG2a; available from ATCC, which are ratanti-murine CD11a antibodies).

Examples of anti-CD18 antibodies include MHM23 (Hildreth et al., supra),M18/2 (IgG2a; Sanches-Madrid et al., J. Exp. Med., 158: 586 [1983]), H52(Fekete et al., J. Clin. Lab Immunol., 31: 145-149 [1990]), Mas191c(Vermot Desroches et al., supra), IOT18 (Vermot Desroches et al.,supra), 60.3 (Taylor et al., Clin. Exp. Immunol., 71: 324-328 [1988]),and 60.1 (Campana et al., Eur. J. Immunol., 16: 537-542 [1986]).

Other examples of suitable LFA-1 antagonists, including antibodies, aredescribed in Hutchings et al., Nature, 348: 639 (1990), WO 91/18011published Nov. 28, 1991, WO 91/16928 published Nov. 14, 1991, WO91/16927 published Nov. 14, 1991, Can. Pat. Appln. 2,008,368 publishedJun. 13, 1991, WO 90/15076 published Dec. 13, 1990, WO 90/10652published Sep. 20, 1990, EP 387,668 published Sep. 19, 1990, EP 379,904published Aug. 1, 1990, EP 346,078 published Dec. 13, 1989, U.S. Pat.No. 5,071,964, U.S. Pat. No. 5,002,869, Australian Pat. Appln. 8815518published Nov. 10, 1988, EP 289,949 published Nov. 9, 1988, and EP303,692 published Feb. 22, 1989.

MODES FOR CARRYING OUT THE INVENTION

1. General Description of the Invention

The current invention is concerned with incorporating a salvage receptorbinding epitope of the Fc region of an IgG into a polypeptide ofinterest so as to increase its circulatory half-life, but so as not tolose its biological activity. This can take place by any means, such asby mutation of the appropriate region in the polypeptide of interest tomimic the Fc region or by incorporating the epitope into a peptide tagthat is then fused to the polypeptide of interest at either end or inthe middle or by DNA or peptide synthesis.

A systematic method for preparing such a polypeptide variant having anincreased in vivo half-life comprises several steps. The first involvesidentifying the sequence and conformation of a salvage receptor bindingepitope on an Fc region of an IgG molecule. Once this epitope isidentified, the sequence of the polypeptide of interest is modified toinclude the sequence and conformation of the identified binding epitope.After the sequence is mutated, the polypeptide variant is tested to seeif it has a longer in vivo half-life than that of the originalpolypeptide, i.e., the polypeptide of interest. If the polypeptidevariant does not have a longer in vivo half-life upon testing, itssequence is further altered to include the sequence and conformation ofthe identified binding epitope. The altered polypeptide is tested forlonger in vivo half-life, and this process is continued until a moleculeis obtained that exhibits a longer in vivo half-life.

The salvage receptor binding epitope being thus incorporated into thepolypeptide of interest is any suitable such epitope as defined above,and its nature will depend, e.g., on the type of polypeptide beingmodified. The transfer is made such that the biological activity of thepolypeptide of interest is maintained, i.e., the transferred portiondoes not adversely affect the conformation of the polypeptide ofinterest or affect its binding to ligands that confers its biologicalactivity. For example, if the polypeptide of interest is an antibody,the salvage receptor binding epitope is not placed so as to interferewith an antigen-binding site of the antibody.

Preferably, the polypeptide of interest contains an Ig domain or Ig-likedomain and the salvage receptor binding epitope is placed so that it islocated within this Ig domain or Ig-like domain. More preferably, theepitope constitutes a region wherein any one or more amino acid residuesfrom one or two loops of the Fc domain are transferred to an analogousposition of the Ig domain or Ig-like domain of the polypeptide ofinterest. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of an Ig or to a Ig-like domain. Alternatively, the epitope istaken from the CH2 domain of the Fc region and transferred to the CLregion or V_(L) region, or both, of an Ig or to an Ig-like domain of thepolypeptide of interest.

For example, for purposes of discussing variants wherein the polypeptideof interest is anti-CD18, reference is made to FIG. 2, which Illustratesthe relevant consensus primary structures of various Igs, i.e., humanIgG1 CH1 domain, human IgG2 CH1 domain, human IgG3 CH1 domain, humanIgG4 CH1 domain, human kappa CL domain, and human lambda CL domain, aswell as the specific sequence for Fab v1b, a preferred anti-CD18 Fabvariant herein. Further, FIG. 2 indicates the residues of Fab v1b thatare of interest and of most importance. In a preferred embodiment, theresidues of importance are those with an asterisk in FIG. 2, i.e., Inone loop of Fab v1b, MIS with a T residue one amino acid C-terminal toMIS, and in another loop of Fab v1b, HQN with a D residue two aminoacids C-terminal to HQN and a K residue one amino acid C-terminal to theD residue.

In one most preferred embodiment, the salvage receptor binding epitopecomprises the sequence (5′ to 3′):

PKNSSMISNTP (SEQ ID NO: 3),

and optionally further comprises a sequence selected from the groupconsisting of HQSLGTQ (SEQ ID NO: 11), HQNLSDG K (SEQ ID NO: 1),HQNISDGK (SEQ ID NO: 2), or VISSHLGQ (SEQ ID NO: 31), particularly wherethe polypeptide of interest is a Fab or (Fab′)₂.

In another most preferred embodiment, the salvage receptor bindingepitope is a polypeptide that is not an Fc containing the sequence(s)(5′to 3′): HQNLSDGK (SEQ ID NO: 1), HQNISDGK (SEQ ID NO: 2), or VISSHLGQ(SEQ ID NO: 31) and the sequence: PKNSSMISNTP (SEQ ID NO: 3). Thisepitope is suitably fused to the polypeptide of interest, and in apreferred aspect is contained on a peptide that is fused to thepolypeptide of interest. Examples of polypeptides of interest suitablefor this purpose include those which will have altered secondary ortertiary structure, with adverse consequences, if the sequence thereofis mutated, such as growth hormone or nerve growth factor.

In one embodiment, the variants can be prepared by recombinant means.Thus, nucleic acid encoding the variant is prepared, placed into areplicable vector and the vector is used to transfect or transformsuitable host cells for expression. The polypeptide variant is producedby culturing the host cells in a culture medium and recovering thepolypeptide variant from the host cell culture. If the polypeptidevariant is being secreted, it is recovered from the culture medium. Inanother embodiment, the polypeptide variant is prepared by altering apolypeptide of interest that is cleared from the kidney and does notcontain an Fc region of an IgG so that it comprises a salvage receptorbinding epitope of an Fc region of an IgG and has an increased in vivohalf-life. The altering step is preferably conducted by Kunkel,site-directed, cassette, or PCR mutagenesis. Kunkel mutagenesis isdescribed, e.g., by Kunkel, Proc. Natl. Acad. Sci. U.S.A., 82: 488-492(1985).

2. Preparation of Polypeptides of Interest and Their Variants

Most of the discussion below pertains to production of the polypeptideof interest or polypeptide variant by culturing cells transformed with avector containing the nucleic acid encoding the polypeptide of interestor polypeptide variant and recovering the polypeptide of interest orvariant from the cell culture. It is further envisioned that thepolypeptide of interest may be produced by homologous recombination, asprovided for in WO 91/06667 published 16 May 1991. Briefly, this methodinvolves transforming primary mammalian cells containing endogenouspolypeptide (e.g., human cells if the desired polypeptide is human) witha construct (i.e., vector) comprising an amplifiable gene (such asdihydrofolate reductase [DHFR] or others discussed below) and at leastone flanking region of a length of at least about 150 bp that ishomologous with a DNA sequence at the locus of the coding region of thegene of the polypeptide of interest to provide amplification of the geneencoding the polypeptide of interest. The amplifiable gene must be at asite that does not interfere with expression of the gene encoding thepolypeptide of interest. The transformation is conducted such that theconstruct becomes homologously integrated into the genome of the primarycells to define an amplifiable region.

Primary cells comprising the construct are then selected for by means ofthe amplifiable gene or other marker present in the construct. Thepresence of the marker gene establishes the presence and integration ofthe construct into the host genome. No further selection of the primarycells need be made, since selection will be made in the second host. Ifdesired, the occurrence of the homologous recombination event can bedetermined by employing PCR and either sequencing the resultingamplified DNA sequences or determining the appropriate length of the PCRfragment when DNA from correct homologous integrants is present andexpanding only those cells containing such fragments. Also if desired,the selected cells may be amplified at this point by stressing the cellswith the appropriate amplifying agent (such as methotrexate if theamplifiable gene is DHFR), so that multiple copies of the target geneare obtained. Preferably, however, the amplification step is notconducted until after the second transformation described below.

After the selection step, DNA portions of the genome, sufficiently largeto include the entire amplifiable region, are isolated from the selectedprimary cells. Secondary mammalian expression host cells are thentransformed with these genomic DNA portions and cloned, and clones areselected that contain the amplifiable region. The amplifiable region isthen amplified by means of an amplifying agent if not already amplifiedin the primary cells. Finally, the secondary expression host cells nowcomprising multiple copies of the amplifiable region containing thepolypeptide of interest are grown so as to express the gene and producethe polypeptide.

A. Isolation of DNA Encoding Polypeptide of Interest

The DNA encoding the polypeptide of interest may be obtained from anycDNA library prepared from tissue believed to possess the mRNA encodingthe polypeptide of interest and to express it at a detectable level. Thegene encoding the polypeptide of interest may also be obtained from agenomic library or by in vitro oligonucleotide synthesis, assuming thecomplete nucleotide or amino acid sequence is known.

Libraries are screened with probes designed to identify the gene ofinterest or the protein encoded by it. For cDNA expression libraries,suitable probes include monoclonal or polyclonal antibodies thatrecognize and specifically bind to the polypeptide of interest;oligonucleotides of about 20-80 bases in length that encode known orsuspected portions of the cDNA encoding the polypeptide of interest fromthe same or different species; and/or complementary or homologous cDNAsor fragments thereof that encode the same or a similar gene. Appropriateprobes for screening genomic DNA libraries include, but are not limitedto, oligonucleotides, cDNAs, or fragments thereof that encode the sameor a similar gene, and/or homologous genomic DNAs or fragments thereof.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures as described in Chapters 10-12 ofSambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1989).

An alternative means to isolate the gene encoding the polypeptide ofinterest is to use PCR methodology as described in Section 14 ofSambrook et al., supra. This method requires the use of oligonucleotideprobes that will hybridize to the polypeptide of interest. Strategiesfor selection of oligonucleotides are described below.

A preferred method of practicing this invention is to use carefullyselected oligonucleotide sequences to screen cDNA libraries from varioustissues.

The oligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The actual nucleotide sequence(s) is usually based on conserved orhighly homologous nucleotide sequences. The oligonucleotides may bedegenerate at one or more positions. The use of degenerateoligonucleotides may be of particular importance where a library isscreened from a species in which preferential codon usage is not known.The oligonucleotide must be labeled such that it can be detected uponhybridization to DNA in the library being screened. The preferred methodof labeling is to use ³²P-labeled ATP with polynucleotide kinase, as iswell known in the art, to radiolabel the oligonucleotide. However, othermethods may be used to label the oligonucleotide, including, but notlimited to, biotinylation or enzyme labeling.

Of particular interest is the nucleic acid encoding the polypeptide ofinterest that encodes a full-length polypeptide. In some preferredembodiments, the nucleic acid sequence includes the polypeptide ofinterest's signal sequence. Nucleic acid having all the protein codingsequence is obtained by screening selected cDNA or genomic librariesusing the deduced amino acid sequence disclosed herein for the firsttime, and, if necessary, using conventional primer extension proceduresas described in Section 7.79 of Sambrook et al., supra, to detectprecursors and processing intermediates of mRNA that may not have beenreverse-transcribed into cDNA.

B. Preparation of Variants of Polypeptide of Interest

The variants of the polypeptide of interest are suitably prepared byintroducing appropriate nucleotide changes as set forth above for the Fcregion into the DNA encoding the polypeptide of interest, or by in vitrosynthesis of the desired polypeptide variant. Such variants include, forexample, deletions from, or insertions or substitutions of, residueswithin the amino acid sequence of the polypeptide of interest so that itcontains the proper epitope and has a longer half-life in serum. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the polypeptide of interest, such aschanging the number or position of glycosylation sites. Moreover, likemost mammalian genes, the polypeptide of interest might be encoded bymulti-exon genes.

For the design of amino acid sequence variants of the polypeptide ofinterest, the location of the mutation site and the nature of themutation will be determined by the specific polypeptide of interestbeing modified. For example, an immunoglobulin or immunoglobulin-likedomain will be initially modified by locating loops that arestructurally similar to the two loops in IgG CH2 that contain thesalvage receptor epitope. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconservative amino acid choices and then with more radical selectionsdepending upon the results achieved, (2) deleting the target residue, or(3) inserting residues of the same or a different class adjacent to thelocated site, or combinations of options 1-3.

A useful method for identification of certain residues or regions of thepolypeptide of interest that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis,” as described by Cunningham andWells, Science, 244: 1081-1085 (1989). Here, a residue or group oftarget residues are identified (e.g., charged residues such as arg, asp,his, lys, and glu) and replaced by a neutral or negatively charged aminoacid (most preferably alanine or polyalanine) to affect the interactionof the amino acids with the surrounding aqueous environment in oroutside the cell. Those domains demonstrating functional sensitivity tothe substitutions then are refined by introducing further or othervariants at or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, tooptimize the performance of a mutation at a given site, alanine scanningor random mutagenesis is conducted at the target codon or region and thevariants produced are screened for increased circulatory half-life.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Contiguous deletions ordinarily are made in even numbers ofresidues, but single or odd numbers of deletions are within the scopehereof. As an example, deletions may be introduced into regions of lowhomology among LFA-1 antibodies which share the most sequence identityto the amino acid sequence of the polypeptide of interest to modify thehalf-life of the polypeptide. Deletions from the polypeptide of interestin areas of substantial homology with one of the binding sites of otherligands will be more likely to modify the biological activity of thepolypeptide of interest more significantly. The number of consecutivedeletions will be selected so as to preserve the tertiary structure ofthe polypeptide of interest in the affected domain, e.g., beta-pleatedsheet or alpha helix.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues. Intra-sequence insertions (i.e.,insertions within the mature polypeptide sequence) may range generallyfrom about 1 to 10 residues, more preferably 1 to 5, most preferably 1to 3. Insertions are preferably made in even numbers of residues, butthis is not required. Examples of insertions include insertions to theinternal portion of the polypeptide of interest, as well as N- orC-terminal fusions with proteins or peptides containing the desiredepitope that will result, upon fusion, in an increased half-life.

A third group of variants are amino acid substitution variants. Thesevariants have at least one amino acid residue in the polypeptidemolecule removed and a different residue inserted in its place. Thesites of greatest interest for substitutional mutagenesis include one ortwo loops in antibodies. Other sites of interest are those in whichparticular residues of the polypeptide obtained from various species areidentical among all animal species of the polypeptide of interest, thisdegree of conservation suggesting importance in achieving biologicalactivity common to these molecules. These sites, especially thosefalling within a sequence of at least three other identically conservedsites, are substituted in a relatively conservative manner. Suchconservative substitutions are shown in Table 1 under the heading ofpreferred substitutions. If such substitutions result in a change inbiological activity, then more substantial changes, denominatedexemplary substitutions in Table 1, or as further described below inreference to amino acid classes, are introduced and the productsscreened. TABLE 1 Original Preferred Residue Exemplary SubstitutionsSubstitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn(N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asnasn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg argIle (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile;val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ileleu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thrThr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val(V) ile; leu; met; phe; ala; norleucine leu

Substantial modifications in function of the polypeptide of interest areaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, lie;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

It may be desirable to inactivate one or more protease cleavage sitesthat are present in the molecule. These sites are identified byinspection of the encoded amino acid sequence, in the case of trypsin,e.g., for an arginyl or lysinyl residue. When protease cleavage sitesare identified, they are rendered inactive to proteolytic cleavage bysubstituting the targeted residue with another residue, preferably abasic residue such as glutamine or a hydrophilic residue such as serine;by deleting the residue; or by inserting a prolyl residue immediatelyafter the residue.

In another embodiment, any methionyl residues other than the startingmethionyl residue of the signal sequence, or any residue located withinabout three residues N- or C-terminal to each such methionyl residue, issubstituted by another residue (preferably in accord with Table 1) ordeleted. Alternatively, about 1-3 residues are inserted adjacent to suchsites.

Any cysteine residues not involved in maintaining the properconformation of the polypeptide of interest also may be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking.

In the first embodiment, nucleic acid molecules encoding amino acidsequence variants of the polypeptide of interest are prepared by avariety of methods known in the art. These methods include, but are notlimited to, preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the polypeptide on whichthe variant herein is based (“polypeptide of interest”).

Oligonucleotide-mediated mutagenesis is a preferred method for preparingsubstitution, deletion, and insertion polypeptide variants herein. Thistechnique is well known in the art as described by Adelman et al., DNA,2: 183 (1983). Briefly, the DNA is altered by hybridizing anoligonucleotide encoding the desired mutation to a DNA template, wherethe template is the single-stranded form of a plasmid or bacteriophagecontaining the unaltered or native DNA sequence of the polypeptide to bevaried. After hybridization, a DNA polymerase is used to synthesize anentire second complementary strand of the template that will thusincorporate the oligonucleotide primer, and will code for the selectedalteration in the DNA.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Natl. Acad. Sci. USA, 75: 5765 (1978).

The DNA template can be generated by those vectors that are eitherderived from bacteriophage M13 vectors (the commercially available M13mp18 and M13 mp19 vectors are suitable), or those vectors that contain asingle-stranded phage origin of replication as described by Viera et al.Meth. Enzymol., 153: 3 (1987). Thus, the DNA that is to be mutated maybe inserted into one of these vectors to generate single-strandedtemplate. Production of the single-stranded template is described inSections 4.21-4.41 of Sambrook et al., supra.

Alternatively, single-stranded DNA template may be generated bydenaturing double-stranded plasmid (or other) DNA using standardtechniques.

For alteration of the original DNA sequence to generate the polypeptidevariants of this invention, the oligonucleotide is hybridized to thesingle-stranded template under suitable hybridization conditions. A DNApolymerizing enzyme, usually the Klenow fragment of DNA polymerase 1, isthen added to synthesize the complementary strand of the template usingthe oligonucleotide as a primer for synthesis. A heteroduplex moleculeis thus formed such that one strand of DNA encodes the mutated form ofthe polypeptide, and the other strand (the original template) encodesthe original, unaltered sequence of the polypeptide. This heteroduplexmolecule is then transformed into a suitable host cell, usually aprokaryote such as E. coli JM101. After the cells are grown, they areplated onto agarose plates and screened using the oligonucleotide primerradiolabeled with ³²P to identify the bacterial colonies that containthe mutated DNA. The mutated region is then removed and placed in anappropriate vector for protein production, generally an expressionvector of the type typically employed for transformation of anappropriate host.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(αS) (which can be obtained from theAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex. Upon addition of DNA polymerase tothis mixture, a strand of DNA identical to the template except for themutated bases is generated. In addition, this new strand of DNA willcontain dCTP-(αS) instead of dCTP, which serves to protect it fromrestriction endonuclease digestion.

After the template strand of the double-stranded heteroduplex is nickedwith an appropriate restriction enzyme, the template strand can bedigested with ExoIII nuclease or another appropriate nuclease past theregion that contains the site(s) to be mutagenized. The reaction is thenstopped to leave a molecule that is only partially single-stranded. Acomplete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101, asdescribed above.

DNA encoding polypeptide mutants with more than one amino acid to besubstituted may be generated in one of several ways. If the amino acidsare located close together in the polypeptide chain, they may be mutatedsimultaneously using one oligonucleotide that codes for all of thedesired amino acid substitutions. If, however, the amino acids arelocated some distance from each other (separated by more than about tenamino acids), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed.

In the first method, a separate oligonucleotide is generated for eachamino acid to be substituted. The oligonucleotides are then annealed tothe single-stranded template DNA simultaneously, and the second strandof DNA that is synthesized from the template will encode all of thedesired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis toproduce the desired mutant. The first round is as described for thesingle mutants: wild-type DNA is used for the template, anoligonucleotide encoding the first desired amino acid substitution(s) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on.

PCR mutagenesis is also suitable for making amino acid variants of thisinvention. While the following discussion refers to DNA, it isunderstood that the technique also finds application with RNA. The PCRtechnique generally refers to the following procedure (see Erlich,supra, the chapter by R. Higuchi, p. 61-70): When small amounts oftemplate DNA are used as starting material in a PCR, primers that differslightly in sequence from the corresponding region in a template DNA canbe used to generate relatively large quantities of a specific DNAfragment that differs from the template sequence only at the positionswhere the primers differ from the template. For introduction of amutation into a plasmid DNA, one of the primers is designed to overlapthe position of the mutation and to contain the mutation; the sequenceof the other primer must be identical to a stretch of sequence of theopposite strand of the plasmid, but this sequence can be locatedanywhere along the plasmid DNA. It is preferred, however, that thesequence of the second primer is located within 200 nucleotides fromthat of the first, such that in the end the entire amplified region ofDNA bounded by the primers can be easily sequenced. PCR amplificationusing a primer pair like the one just described results in a populationof DNA fragments that differ at the position of the mutation specifiedby the primer, and possibly at other positions, as template copying issomewhat error-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer, or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more)-partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide triphosphates and isincluded in the GeneAmp® kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μL. The reaction mixtureis overlaid with 35 μL mineral oil. The reaction mixture is denaturedfor five minutes at 100° C., placed briefly on ice, and then 1 μLThermus aquaticus (Taq) DNA polymerase (5 units/μL, purchased fromPerkin-Elmer Cetus) is added below the mineral oil layer. The reactionmixture is then inserted into a DNA Thermal Cycler (purchased fromPerkin-Elmer Cetus) programmed as follows:

2 min. 55° C.

30 sec. 72° C., then 19 cycles of the following:

-   -   30 sec. 94° C.    -   30 sec. 55° C., and    -   30 sec. 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to the appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., Gene, 34: 315 (1985). Thestarting material is the plasmid (or other vector) comprising the DNA tobe mutated. The codon(s) in the DNA to be mutated are identified. Theremust be a unique restriction endonuclease site on each side of theidentified mutation site(s). If no such restriction sites exist, theymay be generated using the above-described oligonucleotide-mediatedmutagenesis method to introduce them at appropriate locations in theDNA. After the restriction sites have been introduced into the plasmid,the plasmid is cut at these sites to linearize it. A double-strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated DNA sequence.

C. Insertion of Nucleic Acid into Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the polypeptidevariant is inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. Many vectors areavailable, and selection of the appropriate vector will depend on 1)whether it is to be used for DNA amplification or for DNA expression, 2)the size of the nucleic acid to be inserted into the vector, and 3) thehost cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA orexpression of DNA) and the host cell with which it is compatible. Thevector components generally include, but are hot limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

The polypeptide variants of this invention may be produced not onlydirectly, but also as a fusion with a heterologous polypeptide,preferably a signal sequence or other polypeptide having a specificcleavage site at the N-terminus of the mature polypeptide variant. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the DNA that is inserted into the vector. The heterologoussignal sequence selected should be one that is recognized and processed(i.e., cleaved by a signal peptidase) by the host cell. For prokaryotichost cells that do not recognize and process the polypeptide ofinterest's signal sequence, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the groupconsisting of the alkaline phosphatase, penicillinase, Ipp, orheat-stable enterotoxin II leaders. For yeast secretion the original orwild-type signal sequence may be substituted by, e.g., the yeastinvertase leader, yeast alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182 issued 23 Apr. 1991), yeast acid phosphatase leader, mousesalivary amylase leader, carboxypeptidase leader, yeast BAR1 leader,Humicola lanuginosa lipase leader, the C. albicans glucoamylase leader(EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression theoriginal human signal sequence (i.e., the polypeptide presequence thatnormally directs secretion of the native polypeptide of interest fromwhich the variant of interest is derived from human cells in vivo) issatisfactory, although other mammalian signal sequences may be suitable,such as signal sequences from other animal polypeptides and signalsequences from secreted polypeptides of the same or related species, aswell as viral secretory leaders, for example, the herpes simplex gDsignal.

The DNA for such precursor region is ligated in reading frame to DNAencoding the mature polypeptide variant.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 (ATCC37,017), or from other commercially available bacterial vectors such as,e.g., pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1(Promega Biotech, Madison, Wis.), is suitable for most Gram-negativebacteria, the 2μ plasmid origin is suitable for yeast, and various viralorigins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloningvectors in mammalian cells. Generally, the origin of replicationcomponent is not needed for mammalian expression vectors (the SV40origin may typically be used only because it contains the earlypromoter).

Most expression vectors are “shuttle” vectors, i.e., they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of the DNA. However, the recovery of genomic DNA encoding thepolypeptide variant is more complex than that of an exogenouslyreplicated vector because restriction enzyme digestion is required toexcise the DNA.

(iii) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., J. Molec. Appl. Genet., 1: 327[1982]), mycophenolic acid (Mulligan et al., Science, 209: 1422 [1980]),or hygromycin (Sugden et al., Mol. Cell. Biol., 5: 410-413 [1985]). Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thenucleic acid, such as DHFR or thymidine kinase. The mammalian celltransformants are placed under selection pressure that only thetransformants are uniquely adapted to survive by virtue of having takenup the marker. Selection pressure is imposed by culturing thetransformants under conditions in which the concentration of selectionagent in the medium is successively changed, thereby leading toamplification of both the selection gene and the DNA that encodes thepolypeptide variant. Amplification is the process by which genes ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Increased quantities of the polypeptide variant aresynthesized from the amplified DNA. Other examples of amplifiable genesinclude metallothionein-I and -II, preferably primate metallothioneingenes, adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216 (1980). The transformed cells are then exposed toincreased levels of methotrexate. This leads to the synthesis ofmultiple copies of the DHFR gene, and, concomitantly, multiple copies ofother DNA comprising the expression vectors, such as the DNA encodingthe polypeptide variant. This amplification technique can be used withany otherwise suitable host, e.g., ATCC No. CCL61 CHO-K1,notwithstanding the presence of endogenous DHFR if, for example, amutant DHFR gene that is highly resistant to Mtx is employed (EP117,060).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding the polypeptide variant, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 [1979];Kingsman et al., Gene, 7: 141 [1979]; or Tschemper et al., Gene, 10: 157[1980]). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 (Jones, Genetics, 85: 12 [1977]). The presence ofthe trp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC No. 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Bianchi et al.,Curr. Genet., 12: 185 (1987). More recently, an expression system forlarge-scale production of recombinant calf chymosin was reported for K.lactis. Van den Berg, Bio/Technology, 8: 135 (1990). Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed.Fleer et al., Bio/Technology, 9: 968-975 (1991).

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid. Promoters are untranslated sequences located upstream (5′) to thestart codon of a structural gene (generally within about 100 to 1000 bp)that control the transcription and translation of particular nucleicacid sequence, such as the nucleic acid sequence of the polypeptidevariants herein, to which they are operably linked. Such promoterstypically fall into two classes, inducible and constitutive. Induciblepromoters are promoters that initiate increased levels of transcriptionfrom DNA under their control in response to some change in cultureconditions, e.g., the presence or absence of a nutrient or a change intemperature. At this time a large number of promoters recognized by avariety of potential host cells are well known. These promoters areoperably linked to the DNA encoding the polypeptide variant by removingthe promoter from the source DNA by restriction enzyme digestion andinserting the isolated promoter sequence into the vector. The promoterof the polypeptide of interest and many heterologous promoters may beused to direct amplification and/or expression of the DNA. However,heterologous promoters are preferred, as they generally permit greatertranscription and higher yields of recombinantly produced polypeptidevariant as compared to the promoter of the polypeptide of interest.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615[1978]; and Goeddel et al., Nature, 281: 544 [1979]), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057 [1980] and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 [1983]).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding the polypeptide variant(Siebenlist et al., Cell, 20: 269 [1980]) using linkers or adaptors tosupply any required restriction sites. Promoters for use in bacterialsystems also will contain a Shine-Dalgarno (S.D.) sequence operablylinked to the DNA encoding the polypeptide variant.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255: 2073 [1980]) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7: 149 [1968]; and Holland, Biochemistry, 17: 4900[1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657. Yeast enhancers also are advantageouslyused with yeast promoters.

Transcription of polypeptide variant from vectors in mammalian hostcells is controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published,5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand most preferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, and from the promoter normally associated with thepolypeptide variant sequence, provided such promoters are compatiblewith the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature, 273:113 (1978); Mulligan and Berg,Science, 209: 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA, 78: 7398-7402 (1981). The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene, 18: 355-360 (1982). A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.Nature, 295: 503-508 (1982) on expressing cDNA encoding immuneinterferon in monkey cells; Reyes et al., Nature, 297: 598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus; Canaani andBerg, Proc. Natl. Acad. Sci. USA, 79: 5166-5170 (1982) on expression ofthe human interferon β1 gene in cultured mouse and rabbit cells; andGorman et al., Proc. Natl. Acad. Sci. USA, 79: 6777-6781 (1982) onexpression of bacterial CAT sequences in CV-1 monkey kidney cells,chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, andmouse NIH-3T3 cells using the Rous sarcoma virus long terminal repeat asa promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the polypeptide variant of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp, that act on a promoter to increaseits transcription. Enhancers are relatively orientation and positionindependent, having been found 5′ (Laimins et al., Proc. Natl. Acad.Sci. USA, 78: 993 [1981]) and 3′ (Lusky et al., Mol. Cell Bio., 3: 1108[1983]) to the transcription unit, within an intron (Banerji et al.,Cell, 33: 729 [1983]), as well as within the coding sequence itself(Osborne et al., Mol. Cell Bio., 4: 1293 [1984]). Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, Nature, 297: 17-18 (1982) on enhancing elements for activation ofeukaryotic promoters. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the polypeptide-variant-encoding sequence, but ispreferably located at a site 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the polypeptide variant.

(vii) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and relegated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9: 309 (1981) or by the method of Maxam et al., Methods inEnzymology, 65: 499 (1980).

(viii) Transient Expression Vectors

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding the polypeptide variant. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector. Sambrook et al.,supra, pp. 16.17-16.22. Transient expression systems, comprising asuitable expression vector and a host cell, allow for the convenientpositive identification of polypeptide variants encoded by cloned DNAs,as well as for the rapid screening of such polypeptides for desiredbiological or physiological properties. Thus, transient expressionsystems are particularly useful in the invention for purposes ofidentifying polypeptide variants that are biologically active.

(ix) Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the polypeptide variant in recombinant vertebrate cellculture are described in Gething et al., Nature, 293: 620-625 (1981);Mantei et al., Nature, 281: 4046 (1979); EP 117,060; and EP 117,058. Aparticularly useful plasmid for mammalian cell culture production of thepolypeptide variant is pRK5 (EP 307,247) or pSVI6B (WO 91/08291published 13 Jun. 1991). The pRK5 derivative pRK5B (Holmes et al.,Science, 253: 1278-1280119911) is particularly suitable herein for suchexpression.

D. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast, or higher eukaryote cells described above. Suitableprokaryotes for this purpose include eubacteria, such as Gram-negativeor Gram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, andStreptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coliB, E. coli X1776 (ATCC31,537), E. coli DH5σ, and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting. Strain W3110 isone particularly preferred host or parent host because it is a commonhost strain for recombinant DNA product fermentations. Preferably, thehost cell secretes minimal amounts of proteolytic enzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding proteins endogenous to the host, with examples of such hostsincluding E. coli W3110 strain 1A2, which has the complete genotypetonAΔ; E. coli W3110 strain 9E4, which has the complete genotype tonAΔptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the completegenotype tonA ptr3 phoAΔE15 Δ(argF-lac) 169 ΔdegP ΔompT kan′; E. coliW3110 strain 37D6, which has the complete genotype tonA ptr3 phoAΔE15Δ(argF-lac) 169 ΔdegP ΔompT Δrbs7 ilvG kan′; E. coli W3110 strain 40B4,which is strain 37D6 with a non-kanamycin resistant degP deletionmutation; and an E. coli strain having mutant periplasmic proteasedisclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990. Alternatively,in vitro methods of cloning, e.g., PCR or other nucleic acid polymerasereactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forpolypeptide-variant-encoding vectors. Saccharomyces cerevisiae, orcommon baker's yeast, is the most commonly used among lower eukaryotichost microorganisms. However, a number of other genera, species, andstrains are commonly available and useful herein, such asSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., supra) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737 [1983]), K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van denBerg et al., supra), K. thermotolerans, and K. marxianus; yarrowia (EP402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. BasicMicrobiol., 28: 265-278 [1988]); Candida; Trichoderma reesia (EP244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,76: 5259-5263 [1979]); Schwanniomyces such as Schwanniomycesoccidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungisuch as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans(Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 [1983];Tilburn et al., Gene, 26: 205-221 [1983]; Yelton et al., Proc. Natl.Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes,EMBO J., 4: 475-479 [1985]).

Suitable host cells for the production of the polypeptide variant arederived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. See, e.g., Luckow et al., Bio/Technology, 6: 47-55(1988); Miller et al., in Genetic Engineering, Setlow, J. K. et al.,eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al.,Nature, 315: 592-594 (1985). A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the DNA. During incubation of the plant cell culture with A.tumefaciens, the DNA encoding the polypeptide variant is transferred tothe plant cell host such that it is transfected, and will, underappropriate conditions, express the DNA. In addition, regulatory andsignal sequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al., J. Mol. Appl. Gen., 1: 561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. EP321,196 published 21 Jun. 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure in recent years (Tissue Culture, Academic Press, Kruse andPatterson, editors [1973]). Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36; 59 [1977]); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 [1980]); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumorcells (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y.Acad. Sci., 383: 44-68 [1982]); MRC 5 cells; FS4 cells; and a humanhepatoma line (Hep G2).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. Transfection refers to the taking up ofan expression vector by a host cell whether or not any coding sequencesare in fact expressed. Numerous methods of transfection are known to theordinarily skilled artisan, for example, CaPO₄ and electroporation.Successful transfection is generally recognized when any indication ofthe operation of this vector occurs within the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, or electroporation is generally used for prokaryotes orother cells that contain substantial cell-wall barriers. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859published 29 Jun. 1989. In addition, plants may be transfected usingultrasound treatment as described in WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52: 456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described by Axel in U.S. Pat. No. 4,399,216issued 16 Aug. 1983. Transformations into yeast are typically carriedout according to the method of Van Solingen et al., J. Bact., 130: 946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979).However, other methods for introducing DNA into cells, such as bynuclear microinjection, electroporation, bacterial protoplast fusionwith intact cells, or polycations, e.g., polybrene, polyornithine, etc.,may also be used. For various techniques for transforming mammaliancells, see Keown et al., Methods in Enzymology, 185: 527-537 (1990) andMansour et al., Nature, 336: 348-352 (1988).

E. Culturing the Host Cells

Prokaryotic cells used to produce the polypeptide variant of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

The mammalian host cells used to produce the polypeptide variant of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F-10 (Sigma), F-12 (Sigma), Minimal Essential Medium([MEM], Sigma), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium([D-MEM], Sigma), and D-MEM/F-12 (Gibco BRL) are suitable for culturingthe host cells. In addition, any of the media described, for example, inHam and Wallace, Methods in Enzymology, 58: 44 (1979); Barnes and Sato,Anal. Biochem., 102: 255 (1980); U.S. Pat. No. 4,767,704; 4,657,866;4,927,762; 5,122,469; or 4,560,655; U.S. Pat. Re. No. 30,985; WO90/03430; or WO 87/00195 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, aprotinin,and/or epidermal growth factor [EGF]), salts (such as sodium chloride,calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides(such as adenosine and thymidine), antibiotics (such as Gentamycin™drug), trace elements (defined as inorganic compounds usually present atfinal concentrations in the micromolar range), and glucose or anequivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH, andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of in vitro mammalian cell cultures can befound in Mammalian Cell Biotechnology: a Practical Approach, M. Butler,ed. (IRL Press, 1991).

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

F. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77: 5201-5205 [1980]), dot blotting (DNA analysis), orin situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Various labels may be employed, mostcommonly radioisotopes, particularly ³²P. However, other techniques mayalso be employed, such as using biotin-modified nucleotides forintroduction into a polynucleotide. The biotin then serves as the sitefor binding to avidin or antibodies, which may be labeled with a widevariety of labels, such as radionuclides, fluorescers, enzymes, or thelike. Alternatively, antibodies may be employed that can recognizespecific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNAhybrid duplexes or DNA-protein duplexes. The antibodies in turn may belabeled and the assay may be carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75: 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a polypeptide variant as described further in Section 4 below.

G. Purification of Polypeptide

If the variant is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed fragments, is removed,for example, by centrifugation or ultrafiltration; optionally, theprotein may be concentrated with a commercially available proteinconcentration filter, followed by separating the polypeptide variantfrom other impurities by one or more steps selected from immunoaffinitychromatography, ion-exchange column fractionation (e.g., ondiethylaminoethyl (DEAE) or matrices containing carboxymethyl orsulfopropyl groups), chromatography on Blue-Sepharose, CMBlue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose, WGA-Sepharose,Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl, orprotein A Sepharose, SDS-PAGE chromatography, silica chromatography,chromatofocusing, reverse phase HPLC (e.g., silica gel with appendedaliphatic groups), gel filtration using, e.g., Sephadex molecular sieveor size-exclusion chromatography, chromatography on columns thatselectively bind the polypeptide, and ethanol or ammonium sulfateprecipitation.

Recombinant polypeptide variant produced in bacterial culture mayusually be isolated by initial extraction from cell pellets, followed byone or more concentration, salting-out, aqueous ion-exchange, orsize-exclusion chromatography steps. Additionally, the recombinantpolypeptide variant may be purified by affinity chromatography. Finally,HPLC may be employed for final purification steps. Microbial cellsemployed In expression of nucleic acid encoding the polypeptide variantmay be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or through the use of celllysing agents.

A protease inhibitor such as methylsulfonylfluoride (PMSF) may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics may be included to prevent the growth of adventitiouscontaminants.

Within another embodiment, supernatants from systems which secreterecombinant polypeptide variant into culture medium are firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate may be appliedto a suitable purification matrix. For example, a suitable affinitymatrix may comprise a ligand for the protein, a lectin or antibodymolecule bound to a suitable support. Alternatively, an anion-exchangeresin may be employed, for example, a matrix or substrate having pendantDEAE groups. Suitable matrices include acrylamide, agarose, dextran,cellulose, or other types commonly employed in protein purification.Alternatively, a cation-exchange step may be employed. Suitable cationexchangers include various insoluble matrices comprising sulfopropyl orcarboxymethyl groups. Sulfopropyl groups are particularly preferred.

Finally, one or more RP-HPLC steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, may beemployed to further purify a polypeptide variant composition. Some orall of the foregoing purification steps, in various combinations, canalso be employed to provide a homogeneous recombinant polypeptidevariant.

Fermentation of yeast which produce the polypeptide variant as asecreted polypeptide greatly simplifies purification. Secretedrecombinant polypeptide variant resulting from a large-scalefermentation may be purified by methods analogous to those disclosed byUrdal et al., J. Chromatog., 296: 171 (1984). This reference describestwo sequential, RP-HPLC steps for purification of recombinant human IL-2on a preparative HPLC column. Alternatively, techniques such as affinitychromatography, may be utilized to purify the polypeptide variant.

Mammalian polypeptide variant synthesized in recombinant culture ischaracterized by the presence of non-human cell components, includingproteins, in amounts and of a character which depend on the purificationsteps taken to recover the polypeptide variant from culture. Thesecomponents ordinarily will be from yeast, prokaryotic, or non-humanhigher eukaryotic origin and preferably are present in innocuouscontaminant quantities, on the order of less than about 1% by weight.

H. Covalent Modifications of Polypeptide Variants

Covalent modifications of polypeptide variants are included within thescope of this invention. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the variant polypeptide, ifapplicable. Other types of covalent modifications of the polypeptidevariant are introduced into the molecule by reacting targeted amino acidresidues of the polypeptide variant with an organic derivatizing agentthat is capable of reacting with selected side chains or the N- orC-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,o-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 125I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions the deamidatedform of these residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the polypeptide variantincluded within the scope of this invention comprises altering theoriginal glycosylation pattern of the polypeptide variant. By alteringis meant deleting one or more carbohydrate moieties found in thepolypeptide variant, and/or adding one or more glycosylation sites thatare not present in the polypeptide variant.

Glycosylation of polypeptide variants is typically either N-linked orO-linked. N-linked refers to the attachment of the carbohydrate moietyto the side chain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the polypeptide variant isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites). The alteration may also be made by theaddition of, or substitution by, one or more serine or threonineresidues to the sequence of the original polypeptide variant (forO-linked glycosylation sites). For ease, the polypeptide variant aminoacid sequence is preferably altered through changes at the DNA level,particularly by mutating the DNA encoding the polypeptide variant atpreselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) may be made usingmethods described above.

Another means of increasing the number of carbohydrate moieties on thepolypeptide variant is by chemical or enzymatic coupling of glycosidesto the polypeptide variant. These procedures are advantageous in thatthey do not require production of the polypeptide variant in a host cellthat has glycosylation capabilities for N- or O-linked glycosylation.Depending on the coupling mode used, the sugar(s) may be attached to (a)arginine and histidine, (b) free carboxyl groups, (c) free sulfhydrylgroups such as those of cysteine, (d) free hydroxyl groups such as thoseof serine, threonine, or hydroxyproline, (e) aromatic residues such asthose of phenylalanine, tyrosine, or tryptophan, or (f) the amide groupof glutamine. These methods are described in WO 87/05330 published 11Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp.259-306 (1981).

Removal of any carbohydrate moieties present on the polypeptide variantmay be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the polypeptide variant to thecompound trifluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide variant intact. Chemical deglycosylation isdescribed by Hakimuddin, et al., Arch. Biochem. Biophys., 259: 52 (1987)and by Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavageof carbohydrate moieties on polypeptide variants can be achieved by theuse of a variety of endo- and exo-glycosidases as described by Thotakuraet al., Meth. Enzymol., 138: 350 (1987).

Glycosylation at potential glycosylation sites may be prevented by theuse of the compound tunicamycin as described by Duskin et al., J. Biol.Chem., 257: 3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the polypeptide variantcomprises linking the polypeptide variant to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

3. Therapeutic Compositions: Administration of Variant

Uses of anti-CD18 variants include anti-Mac1/anti-neutrophil as well asanti-LFA-1 applications. If the polypeptide variant acts as an antibodyit may optionally be fused to a second polypeptide and the antibody orfusion thereof may be used to isolate and purify the protein to which itbinds from a source such as a CD11 or CD18 antigen. In anotherembodiment, the invention provides a method for detecting CD11a or CD18in vitro or in vivo comprising contacting the anti-CD11a or CD18antibody fragment variant herein with a sample, especially a serumsample, suspected of containing the CD11a or CD18 and detecting ifbinding has occurred.

The polypeptide variant herein is also suitably used in quantitativediagnostic assays as a standard or control against which samplescontaining unknown quantities of the polypeptide variant may beprepared.

Therapeutic formulations of the polypeptide variant for its particularindication are prepared for storage by mixing the polypeptide varianthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, 16th edition, Oslo, A., Ed., [1980]), in theform of lyophilized cake or aqueous solutions. Acceptable carriers,excipients, or stabilizers are non-toxic to recipients at the dosagesand concentrations employed, and include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counter-ions such as sodium; and/or non-ionicsurfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

Typically, the polypeptide variant used in the method of this inventionis formulated by mixing it at ambient temperature at the appropriate pH,and at the desired degree of purity, with physiologically acceptablecarriers, i.e., carriers that are non-toxic to recipients at the dosagesand concentrations employed. The pH of the formulation depends mainly onthe particular use and the concentration of the variant, but preferablyranges anywhere from about 3 to about 8. Formulation in a buffer at pHabout 5-8 is one suitable embodiment.

The polypeptide variant for use herein is preferably sterile. Sterilityis readily accomplished by sterile filtration through (0.2 micron)membranes. The polypeptide variant ordinarily will be stored as anaqueous solution, although lyophilized formulations for reconstitutionare acceptable.

The variant composition will be formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of polypeptide variant to beadministered will be governed by such considerations, and, for an LFA-1antagonist variant, is the minimum amount necessary to prevent,ameliorate, or treat the LFA-1-mediated disorder, including treatingrheumatoid arthritis, reducing inflammatory responses, inducingtolerance of immunostimulants, preventing an immune response that wouldresult in rejection of a graft by a host or vice-versa, or prolongingsurvival of a transplanted graft. The amount of the variant ispreferably below the amount that is toxic to the host or renders thehost significantly more susceptible to infections.

As a general proposition, the initial pharmaceutically effective amountof the LFA-1 antagonist variant administered parenterally per dose willbe in the range of about 0.1 to 20 mg/kg of patient body weight per day,with the typical initial range of LFA-1 antagonist variant used beingabout 0.3 to 15 mg/kg/day.

As noted above, however, these suggested amounts of LFA-1 antagonistvariant are subject to a great deal of therapeutic discretion. The keyfactor in selecting an appropriate dose and scheduling is the resultobtained, as indicated above. For example, relatively higher doses maybe needed initially for the treatment of ongoing and acute graftrejection, or at a later stage for the treatment of acute rejection,which is characterized by a sudden decline in graft function.

Where the subsequent dosing is less than 100% of initial dosing, it iscalculated on the basis of daily dosing. Thus, for example, if thedosing regimen consists of daily injections of 2 mg/kg/day for 2 weeksfollowed by a biweekly dose of 0.5 mg/kg/day for 99 days, this wouldamount to a subsequent dose of about 1.8% of the initial dose,calculated on a daily basis (i.e., 2/day/100%=0.5/14 days/x %, x=−1.8%).Preferably, the subsequent dosing is less than about 50%, morepreferably, less than about 25%, more preferably, less than about 10%,still more preferably, less than about 5%, and most preferably, lessthan about 2% of the initial dosing of LFA-1 antagonist variant.

To obtain the most efficacious results for the LFA-1 antagonist variant,depending on the disorder, the initial dosing is given as close to thefirst sign, diagnosis, appearance, or occurrence of the disorder aspossible or during remissions of autoimmune disorders. Preferably theinitial dosing begins before exposure to antigen, as in the case withtransplanted grafts. Furthermore, when the initial dosing is prior to orsubstantially contemporaneous with exposure to antigen, it is preferredthat the subsequent dosing is carried out for a longer period of timethan the initial dosing, particularly for transplants, and that it be acontinuous intermittent maintenance dose that need not be continuous forthe life of the patient.

The preferred scheduling for the LFA-1 antagonist variant is that theinitial dosing (i.e., administered before or at the time of theundesired immune response at a dose administered no less frequently thandaily up to and including continuously by infusion) and the subsequentdosing is a dose administered periodically no more than about once aweek. More preferably, depending on the specific disorder, andparticularly for transplantation, the initial daily dosing isadministered for at least about one week, preferably at least about 2weeks, after the exposure to antigen, e.g., graft, or initiation of anacute immune response (as in autoimmune disorders), and the subsequentdosing is administered no more than once biweekly (preferably oncebiweekly) for at least about 5 weeks, preferably for at least about 10weeks, after the initial dosing is terminated.

In another preferred embodiment, particularly if the antagonist variantis a Fab or (Fab′)₂ of anti-CD11a or anti-CD18 antibodies, initialdosing terminates from about 1 day to 4 weeks after transplantation hasoccurred, more preferably from about 1 week to 3 weeks, more preferablyfrom about 2 weeks to 3 weeks, and commences from about 1 week beforetransplantation occurs up to about simultaneously with thetransplantation.

The polypeptide variant is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration (including perfusing or otherwisecontacting the graft with the antagonist before transplantation).Parenteral infusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, the LFA-1antagonist variant is suitably administered by pulse infusion,particularly with declining doses of the LFA-1 antagonist variant.Preferably the dosing of such variant is given by injections, mostpreferably intravenous or subcutaneous injections, depending in part onwhether the administration is brief or chronic.

The polypeptide variant herein need not be, but is optionally formulatedwith one or more agents currently used to prevent or treat the disorderin question. For example, in rheumatoid arthritis, an LFA-1 antagonistvariant may be given in conjunction with a glucocorticosteroid. Inaddition, T cell receptor peptide therapy is suitably an adjunct therapyto prevent clinical signs of autoimmune encephalomyelitis. Offner etal., Science, 251: 430-432 (1991). For transplants, the LFA-1 antagonistvariant may be administered concurrently with or separate from animmunosuppressive agent as defined above, e.g., cyclosporin A, tomodulate the immunosuppressant effect. The effective amount of suchother agents depends on the amount of LFA-1 antagonist variant presentin the formulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as used hereinbefore or about from 1 to 99% of theheretofore employed dosages.

The various autoimmune disorders described above are treated with LFA-1antagonist variants in such a fashion as to induce immune tolerance tothe self antigen under attack as a result of the disorder. In thisregard, autoimmune disorders resemble host versus graft rejection andare treated with LFA-1 antagonist variants in analogous fashion.However, in these disorders the patient is already mounting an immuneresponse to the target antigen, unlike the case with transplants priorto grafting. Thus, it is desirable to first induce and maintain atransient state of immunosuppression by conventional methods in suchpatients, e.g., by the conventional use of cyclosporin A or otherconventional immunosuppressive agents (alone or together with LFA-1antagonist variant), or to monitor the patient until the occurrence of aperiod of remission (an absence or substantial lessening of pathologicalor functional indicia of the autoimmune response).

Preferably, transient immunosuppression is induced by T cell depletionusing conventional therapy. This is then followed by the administrationof the LFA-1 antagonist variant in order to prevent rebound when theimmunosuppressive inducing agent is withdrawn or when remissionotherwise would abrogate. Alternatively, the remission patient'scondition is closely monitored for signs of flare, and immediately uponthe initial functional or biochemical appearance of flare the initialdosing regimen is started and continued until the flare subsides. TheLFA-1 antagonist variant administration during this period constitutesthe initial dose described elsewhere herein.

In the case of autoimmune disorders the initial dose will extend aboutfrom 1 week to 16 weeks. Thereafter, the lower dose maintenance regimenof LFA-1 antagonist variant is administered in substantially the samefashion as set forth herein for the amelioration of graft or hostrejection, although in some instances it is desirable to extend thesubsequent or sustaining dose for lengthier periods than with grafts. Inan embodiment of this invention, if an antigen or a compositioncontaining the antigen is known to be responsible for the autoimmuneresponse then the antigen is administered to the patient (optionallywith IL-1 and/or gamma interferon) after the initial LFA-1 antagonistvariant dose and the antagonist variant dose maintained thereafter inorder to suppress the regeneration of an autoimmune response against theantigen while minimally immunosuppressing the patient's response toother antigens.

The patient optimally will be isolated, preferably in an asepticenvironment such as is currently used in transplant practice, at thetime of initial treatment with LFA-1 antagonist variant. The patientshould be free of any infection. It is not necessary to sustain theseconditions during the maintenance dose, and in fact this is one of theadvantages of this invention, i.e., that the patient is able to mount asubstantially normal immune response to ambient antigens (other than thegraft or self antigen) while being treated with the maintenance dosing.

The invention herein is particularly amenable to prolonging survival andincreasing tolerance of transplanted grafts. The transplants areoptionally functionally monitored systematically during the criticalpostoperative period (the first three months) using any suitableprocedure. One such procedure is radionuclide intravenous angiographyusing 99Tcm-pertechnetate, as described by Thomsen et al., Acta Radiol.,29: 138-140 (1988). In addition, the method herein is amenable tosimultaneous, multiple organ perfusion and transplantation.Toledo-Pereyra and MacKenzie, Am. Surg., 46: 161-164 (1980).

In some instances, it is desirable to modify the surface of the graft soas to provide positively or negatively charged groups, as by using asuitable amino acid or polymer or by attaching a physiologicallyacceptable source of charged functional groups. For example, anegatively charged surface is appropriate for blood vessels to diminishblood clotting. It also is desirable in certain circumstances to renderthe surface hydrophobic or hydrophilic by coupling, e.g., phenylalanine,serine or lysine to the surface. An immunosuppressive agent particularlyeffective for these surface modifications is glutaraldehyde.

As mentioned above, before transplantation an effective amount of theLFA-1 antagonist variant is optionally administered to induce toleranceof the graft. The same dose and schedule as used for initialpost-transplantation may be employed. Furthermore, prior totransplantation the graft is optionally contacted with a TGF-βcomposition as described in U.S. Pat. No. 5,135,915, the disclosure ofwhich is incorporated by reference. Briefly, the contact suitablyInvolves incubating or perfusing the graft with the composition orapplying the composition to one or more surfaces of the graft. Thetreatment generally takes place for at least one minute, and preferablyfrom 1 minute to 72 hours, and more preferably from 2 minutes to 24hours, depending on such factors as the concentration of TGF-β in theformulation, the graft to be treated, and the particular type offormulation. Also as noted, the graft is simultaneously or separatelyperfused with LFA-1 antagonist variant. Perfusion is accomplished by anysuitable procedure. For example, an organ can be perfused via a devicethat provides a constant pressure of perfusion having a pressureregulator and overflow situated between a pump and the organ, asdescribed by DD 213,134 published Sep. 5, 1984. Alternatively, the organis placed in a hyperbaric chamber via a sealing door and perfusate isdelivered to the chamber by a pump that draws the fluid from thereservoir while spent perfusate is returned to the reservoir by a valve,as described in EP 125,847 published Nov. 21, 1984.

After the graft is treated, it is suitably stored for prolonged periodsof time or is used immediately in the transplant procedure. Storage lifecan be enhanced as described above by using a blood substitute in theformulation (e.g., perfluorochemical emulsion), or by perfusing thegraft with a formulation of a TGF-β containing chilled isotonic agentand anticoagulant followed by glycerol to allow for freezing of removedorgans with no destruction of the cells, as described in JP 60061501published Apr. 9, 1985. In addition, the organs can be preserved withknown perfusion fluids (containing TGF-β and/or LFA-1 antagonist asnoted) while the organs are cooled to freezing temperatures, to preservethe organ semi-permanently without cell necrocytosis, as described byU.S. Pat. Nos. 4,462,215 and 4,494,385.

Respecting cardiac transplants specifically, Parent et al., Cryobiology,18: 571-576 (1981) reports that cold coronary perfusion prior totransplantation at 5° C. increases protection of the homograft duringthe initial period of implantation. Any of these procedures, or others,are within the scope of this invention if deemed necessary for graftpreservation.

Before transplantation, the graft is preferably washed free of the TGF-βcomposition, as by soaking it in a physiological saline solution or byother means appropriate for this purpose. It is not desirable to removethe LFA-1 antagonist variant prior to transplantation.

Also, prior to transplantation, the host is optionally given one or moredonor-specific blood transfusions to aid in graft survival. Analternative procedure is to subject the host to total lymphoidirradiation prior to or after the transplantation operation. Any otherpre-transplant procedures that would be beneficial to the particulartransplant recipient can be performed as part of the method of thisinvention.

4. Antibody Preparation (where Variant is Antibody-derived)

(i) Starting Materials and Methods

Immunoglobulins (Ig) and certain variants thereof are known and manyhave been prepared in recombinant cell culture. For example, see U.S.Pat. No. 4,745,055; EP 256,654; EP 120,694; EP 125,023; EP 255,694; EP266,663; WO 88/03559; Faulkner et al., Nature, 298: 286 (1982);Morrison, J. Immun., 123: 793 (1979); Koehler et al., Proc. Natl. Acad.Sci. USA, 77: 2197 (1980); Raso et al., Cancer Res., 41: 2073 (1981);Morrison et al., Ann. Rev. Immunol., 2: 239 (1984); Morrison, Science,229: 1202 (1985); and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984). Reassorted immunoglobulin chains are also known. See, forexample, U.S. Pat. No. 4,444,878; WO 88/03565; and EP 68,763 andreferences cited therein. The immunoglobulin moiety in the polypeptidevariants of the present invention may be obtained from IgG-1, IgG-2,IgG-3, or IgG-4 subtypes, IgA, IgE, IgD, or IgM, but preferably fromIgG-1 or IgG-3.

(ii) Polyclonal Antibodies

Polyclonal antibodies are generally raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═CFNR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining 1 mg or 1 μg of the peptide or conjugate (forrabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites. Onemonth later the animals are boosted with ⅕ to 1/10 the original amountof peptide or conjugate in Freund's complete adjuvant by subcutaneousinjection at multiple sites. Seven to 14 days later the animals are bledand the serum is assayed for antibody titer. Animals are boosted untilthe titer plateaus. Preferably, the animal is boosted with the conjugateof the same antigen, but conjugated to a different protein and/orthrough a different cross-linking reagent. Conjugates also can be madein recombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

(iii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler and Milstein, Nature, 256: 495 (1975),or may be made by recombinant DNA methods (Cabilly et al., supra).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 [Academic Press, 1986]).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133: 3001 [1984]; Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 [MarcelDekker, Inc., New York, 1987]).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson and Pollard, Anal.Biochem., 107: 220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown In vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348: 552-554 (1990), using theproper antigen such as CD11a, CD18, IgE, or HER-2 to select for asuitable antibody or antibody fragment. Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991)describe the isolation of murine and human antibodies, respectively,using phage libraries. Subsequent publications describe the productionof high affinity (nM range) human antibodies by chain shuffling (Mark etal., Bio/Technology, 10: 779-783 [1992]), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 [1993]). Thus, these techniques are viable alternatives totraditional monoclonal antibody hybridoma techniques for isolation of“monoclonal” antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (Cabilly et al., supra; Morrison, etal., Proc. Nat. Acad. Sci., 81: 6851 [1984]), or by covalently joiningto the immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the variants herein derived from antibodiestypically will be labeled with a detectable moiety. The detectablemoiety can be any one which is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; radioactive isotopic labels, such as, e.g.,¹²⁵I, ³²P, ¹⁴C, or ³H; or an enzyme, such as alkaline phosphatase,beta-galactosidase, or horseradish peroxidase.

Any method known in the art for separately conjugating the polypeptidevariant to the detectable moiety may be employed, including thosemethods described by Hunter et al., Nature, 144: 945 (1962); David etal., Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem, and Cytochem., 30: 407 (1982).

(iv) Humanized and Human Antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321: 522-525 [1986]; Riechmann et al., Nature,332: 323-327 [1988]; Verhoeyen et al., Science, 239: 1534-1536 [1988]),by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly et al., supra), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151: 2296 [1993]; Chothia and Lesk, J. Mol. Biol., 196: 901[1987]). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89: 4285 [1992]; Presta et al., J. Immnol., 151: 2623 [1993]).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., Proc. Natl. Aced. Sci. USA, 90: 2551-255 (1993);Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Yearin Immuno., 7: 33 (1993). Human antibodies can also be produced inphage-display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381[1991]; Marks et al., J. Mol. Biol., 222: 581 [1991]).

(v) Bispecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different antigens. Bispecific antibodiescan be derived from full length antibodies or antibody fragments (e.g.F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein and Cuello,Nature, 305: 537-539 [1983]). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, published 13 May 1993, and inTraunecker et al., EMBO J., 10: 3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy, chain with a firstbinding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in WO 94/04690published Mar. 3, 1994. For further details of generating bispecificantibodies see, for example, Suresh et al., Methods in Enzymology,121:210 (1986).

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the BsAb. The BsAbs produced can be used as agents for theselective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized BsAb F(ab′)₂ molecule. Each Fab′fragment was separately secreted from E. coli and subjected to directedchemical coupling in vitro to form the BsAb. The BsAb thus formed wasable to bind to cells overexpressing the HER2 receptor and normal humanT cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets. See also Rodrigues etal., Int. J. Cancers, (Suppl.) 7: 45-50 (1992).

Various techniques for making and isolating BsAb fragments directly fromrecombinant cell culture have also been described. For example,bispecific F(ab′)₂ heterodimers have been produced using leucinezippers. Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992). Theleucine zipper peptides from the Fos and Jun proteins were linked to theFab′ portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The “diabody” technologydescribed by Hollinger et al., Proc. Natl. Aced. Sci. (USA), 90:6444-6448 (1993) has provided an alternative mechanism for making BsAbfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingBsAb fragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152: 5368 (1994). Theseresearchers designed an antibody which comprised the V_(H) and V_(L)domains of a first antibody joined by a 25-amino-acid-residue linker tothe V_(H) and V_(L) domains of a second antibody. The refolded moleculebound to fluorescein and the T-cell receptor and redirected the lysis ofhuman tumor cells that had fluorescein covalently linked to theirsurface.

5. Uses of Antibody Variants

Variant antibodies are useful in diagnostic assays for an antigen ofinterest, e.g., its production in specific cells, tissues, or serum. Thevariant antibodies are labeled In the same fashion as described aboveand/or are immobilized on an insoluble matrix. In one embodiment of anantigen-binding assay, an antibody composition that binds to the antigenis immobilized on an insoluble matrix, the test sample is contacted withthe immobilized variant antibody composition to adsorb the antigen, andthen the Immobilized antigen is contacted with variant antibodiesspecific for the antigen, as determined by unique labels such asdiscrete fluorophores or the like. By determining the presence and/oramount of each unique label, the relative proportion and amount of theantigen can be determined.

The variant antibodies of this invention are also useful in passivelyimmunizing patients.

The variant antibodies also are useful for the affinity purification ofan antigen of interest from recombinant cell culture or natural sources.

Suitable diagnostic assays for an antigen and its variant antibodies arewell known per se. In addition to the bioassays described in theexamples below wherein the candidate variant is tested to see if it hasappropriate biological activity and increased half-life, competitive,sandwich and steric inhibition immunoassay techniques are useful. Thecompetitive and sandwich methods employ a phase-separation step as anintegral part of the method while steric inhibition assays are conductedin a single reaction mixture. Fundamentally, the same procedures areused for the assay of the antigen and for substances that bind theantigen, although certain methods will be favored depending upon themolecular weight of the substance being assayed. Therefore, thesubstance to be tested is referred to herein as an analyte, irrespectiveof its status otherwise as an antigen or variant antibody, and proteinsthat bind to the analyte are denominated binding partners, whether theybe antibodies, cell-surface receptors, or antigens.

Analytical methods for the antigen or its variant antibodies all use oneor more of the following reagents: labeled analyte analogue, immobilizedanalyte analogue, labeled binding partner, immobilized binding partner,and steric conjugates. The labeled reagents also are known as “tracers.”

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the binding partner from any analytethat remains free in solution. This conventionally is accomplished byeither insolubilizing the binding partner or analyte analogue before theassay procedure, as by adsorption to a water-insoluble matrix or surface(Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (forexample, using glutaraldehyde cross-linking), or by insolubilizing thepartner or analogue afterward, e.g., by immunoprecipitation.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer analogue to competewith the test sample analyte for a limited number of binding sites on acommon binding partner. The binding partner generally is insolubilizedbefore or after the competition and then the tracer and analyte bound tothe binding partner are separated from the unbound tracer and analyte.This separation is accomplished by decanting (where the binding partnerwas preinsolubilized) or by centrifuging (where the binding partner wasprecipitated after the competitive reaction). The amount of test sampleanalyte is inversely proportional to the amount of bound tracer asmeasured by the amount of marker substance. Dose-response curves withknown amounts of analyte are prepared and compared with the test resultsto quantitatively determine the amount of analyte present in the testsample. These assays are called ELISA systems when enzymes are used asthe detectable markers.

Another species of competitive assay, called a “homogeneous” assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theanalyte is prepared and used such that when anti-analyte binds to theanalyte the presence of the anti-analyte modifies the enzyme activity.In this case, the antigen or its immunologically active fragments areconjugated with a bifunctional organic bridge to an enzyme such asperoxidase. Conjugates are selected for use with anti-polypeptide sothat binding of the anti-polypeptide inhibits or potentiates the enzymeactivity of the label. This method per se is widely practiced under thename of EMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small analyte so that antibody tohapten substantially is unable to bind the conjugate at the same time asanti-analyte. Under this assay procedure the analyte present in the testsample will bind anti-analyte, thereby allowing anti-hapten to bind theconjugate, resulting in a change in the character of the conjugatehapten, e.g., a change in fluorescence when the hapten is a fluorophore.

Sandwich assays particularly are useful for the determination ofpolypeptide variants or polypeptide variant antibodies. In sequentialsandwich assays an immobilized binding partner is used to adsorb testsample analyte, the test sample is removed as by washing, the boundanalyte is used to adsorb labeled binding partner, and bound material isthen separated from residual tracer. The amount of bound tracer isdirectly proportional to test sample analyte. In “simultaneous” sandwichassays the test sample is not separated before adding the labeledbinding partner. A sequential sandwich assay using a monoclonal antibodyas one antibody and a polyclonal antibody as the other is useful intesting samples for antigen activity.

The foregoing are merely exemplary diagnostic assays for the polypeptidevariant and variant antibodies. Other methods now or hereafter developedfor the determination of these analytes are included within the scopehereof, including the bioassays described above.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

EXAMPLE 1

Methods

Plasmid Construction

The template plasmid, pH52, used for constructing the Fabs (hereafterreferred to as Fab) employed in this example was derived from theplasmid pB0475 described by Cunningham et al., Science 243: 1330-1336(1989). Two BamHI sites flanking the F1 origin were removed from pB0475and DNA coding for anti-CD18 Fab H52, version OZ (Eigenbrot et al.,Proteins, 18: 49-62 [1994]) was substituted for DNA coding for humangrowth hormone using the EcoRV and SphI sites. Hence, pH52 contains DNAcoding for anti-CD18 Fab H52 (version OZ), the STII signal peptides ofthe light and heavy chain, the alkaline phosphatase promoter region, anM13 helper phage region, and ampicillin-resistance. Fab variants wereconstructed by Kunkel mutagenesis (Kunkel, Proc. Natl. Acad. Sci.U.S.A., 82: 488-492 [1985]) of pH52 using the followingoligonucleotides:

oligo V1A 5′ GTGACCGTGCCTCACCAGAGCTTGGGCAC3′ (SEQ ID NO: 12)

changes Ser195-Ser196 to His195-Gln196

oligo V1B 5′ TGGCACCCTCCCCTAAGAACTCGAGCATGATCAGCAACACACCGGCCCTGGGC3′(SEQ ID NO: 13)

changes Ser127-Ser-Lys-Ser-Thr-Ser-Gly-Gly-Thr-Ala-Ala139 (SEQ ID NO:14)

to Ser 27-Pro-Lys-Asn-Ser-Ser-Met-Ile-Ser-Asn-Thr-Pro-Ala139 (SEQ ID NO:15)

oligo V1C₅′ TGGCACCCTCCAAATCGAGCATCACAGCGGCCCT3′ (SEQ ID NO: 16)

changes Ser127-Ser-Lys-Ser-Thr-Ser-Gly-Gly-Thr137 (SEQ ID NO: 17)

to Ser127-Lys-Ser-Ser-ile-Thr137 (SEQ ID NO: 18)

oligo V2 5′ TGGTGACCGTGATCTCGAGCCACTTGGGCCAGCAGACCTACATC3′ (SEQ ID NO:19)

changes Val193-Pro-Ser-Ser-Ser-Leu-Gly-Thr-Gln203 (SEQ ID NO: 20)

to Val93-Ile-Ser-Ser-His-Leu-Gly-Gln-Gln203 (SEQ ID NO: 21)

Amino acid residue numbers are according to the numbering systemdescribed in Kabat et al., supra, NIH Publ. No. 91-3242, Vol. 1, pages647-669 (1991).

Fab v1 incorporated oligos V1A and V1C; Fab v1b incorporated oligos V1Aand V1B; Fab v2 incorporated oligo V2. Plasmids coding for Fab v1, Fabv1b, and Fab v2 were selected and the DNA sequences checked usingdideoxynucleotide sequencing (Sequenase™ protocol, United StatesBiochemical). F(ab′)₂ constructs were made by inserting DNA coding forthe IgG1 hinge region followed by a ‘leucine zipper’ at the C-terminusof the H52 heavy constant domain. The inserted amino acid sequence was:

CPPCPAPELLGGRMKQLEDKVEELLSKNYHLENEVARLKKLVGER (SEQ ID NO: 22).

Another set of Fab versions is based on Fab v1b, i.e., the variant whichshowed longer half life, using the following oligonucleotides:

oligo V1D 5′ TCGAGCATGATCTCTAGAACACCGGCCC3′ (SEQ ID NO: 23)

changes Asn136 to Arg136

oligo V1E 5′ GCCTCACCAGAACCTAGGCACCAAGACCTACATCTG3′ (SEQ ID NO: 24)

changes Ser197 to Asn197 and Gln203 to Lys203

oligo V1F 5′ GCCTCACCAGdAACTTAAGCGACGGAAAGACCTACATCTGC3′ (SEQ ID NO: 25)

changes Gln196-Ser-Leu-Gly-Thr-Gln-Thr204 (SEQ ID NO: 26)

to Gln196-Asn-Leu-Ser-Asp-Gly-Lys-Thr204 (SEQ ID NO: 27)

oligo V1G 5′ GCCTCACCAGAATATTACAGATGGCAAGACCTACATCTGC3′ (SEQ ID NO: 28)

changes Gln196-Ser-Leu-Gly-Thr-Gln-Thr204 (SEQ ID NO: 29)

to Gln196-Asn-Ile-Ser-Asp-Gly-Lys-Thr204 (SEQ ID NO: 30)

Fab v3 incorporates oligo V1D; Fab v4 incorporates oligo V1E; Fab v5incorporates oligo V1F; and Fab v6 incorporates oligo V1G.

Expression of DNA Encoding the Variants

For each variant, plasmid DNA was transformed into E. coli. Thetransformants were then plated on Luria Broth (LB) plates containing 5μg/mL carbenicillin and incubated at 37° C. overnight. A single colonywas inoculated into 5 mL [LB+5 μg/mL carbenicillin] and grown for 6-7hours at 37° C. The 5-mL culture was then added to 500 mL AP5 minimalmedia in a 2-L baffled flask and grown for 16 hours at 37° C.

AP5 minimal media is made as follows: Per 1 liter is added 1.5 g glucose(Sigma™ G-7021), 2.2 g casamino acids technical (Difco™ 0231-01-0), 0.3g yeast extract certified (Difco™ 0127-01-7), 0.19 g MgSO₄ anhydrous or0.394 g MgSO₄.7H₂O (Sigma™ M2773), 1.07 g ammonium chloride (Sigma™A9434), 0.075 g KCl (Sigma™ P5405), 4.09 g NaCl (Sigma™ S3014), 120.0 mLof 1M triethanolamine pH 7.4, qs to 1.0 L Super-Q™ Water, as well as 1 Mtriethanolamine pH 7.4 consisting of 133.21 mL triethanolamine, Liquid(Sigma™ T1377) and 950 mL Super-Q™ Water, pH to 7.4 with HCl(Mallinckrodt™ 2612), qs to 1.0 L Super-Q™ Water. This is filteredthrough a 0.1 μm Sealkleen™ filter and stored at 2-8° C. The expirationperiod is 6 months.

The cells were spun in a 1-L centrifuge bottle at 3000 rpm for 30minutes, the supernatant was decanted and the pelleted cells were frozenfor 1 hour. The pellet was resuspended in 10 mL of cold TE buffer (10 mMTRIS, 1 mM EDTA, pH 7.6) with 100 μL 0.1 M benzamidine (Sigma) added.The resuspended pellet was agitated on ice for 1 hour, spun at 18,000rpm for 15 minutes, and the supernatant decanted and held on ice.

The supernatant was then passed over a Protein G-Sepharose™ Fast Flow(Pharmacia) column [0.5 mL bed volume] previously equilibrated bypassing 10 mL TE buffer through the column. The column was then washedwith 10 mL TE buffer, and the Fab eluted with 2.5 mL 100 mM acetic acid,pH 2.8, into a tube containing 0.5 mL TRIS, pH 8.0. The eluant wasconcentrated in a Centricon-30™ (Amicon) centrifuge to 0.5 mL, 2 mLphosphate-buffered saline was added to concentrated eluant, and theresulting mixture was re-concentrated to 0.5 mL. SDS-PAGE gels were runto ascertain that protein had been produced.

Analytical Methods Used During Purification Procedure of Anti-CD11/CD18Fab Variants and F(ab′)₂ Antibody Fragment

SDS polyacrylamide gel electrophoresis (SDS-PAGE) and two different highperformance liquid chromatography (HPLC) methods were used to analyzethe products obtained in each step of the purification process. The HPLCmethods used include reverse-phase chromatography and cation-exchangechromatography, which were performed on a WATERS™ HPLC system.

Reverse-phase chromatography was carried out on a reverse-phase PLRP-S™4.6×50 mm column, 8-mm particle size (Polymer Laboratories, Shropshire,UK), maintained at 50° C. The proteins were eluted using an increasinglinear gradient from 31% B to 41% B. Buffer A contained 0.1%trifluoroacetic acid in deionized water, and Buffer B contained 0.1%trifluoroacetic acid in HPLC-grade acetonitrile. The flow rate wasmaintained at 2 mL/min, and the protein profile was monitored at 214 nm.

Analysis by cation-exchange chromatography was carried out on aBakerbond carboxy-sulfon (CSX)™ 50×4.6 mm column (J. T. BakerPhillipsburg, N.J.), maintained at 55° C. The proteins were eluted usingan increasing linear gradient from pH 6.0 to pH 8.0 at a flow rate of 2mL/min using a detection wavelength of 280 nm. Buffer A contained 16 mMeach of HEPES/PIPES/MES, pH 6.0, and Buffer B contained 16 mM each ofHEPES/PIPES/MES, pH 8.0. For the separation of the different Fabvariants, a linear gradient was run for 22 min from 25% B to 56% B. Forthe separation of the Zipper-F(ab′)2 and F(ab′)2 antibody fragments, thelinear gradient was run from 40% B to 100% B in 22 minutes.

SDS-PAGE analysis was carried out on precast Novex™ gels (Novex, SanDiego, Calif.). The proteins were stained using the Morrissey silverstain method. Morrissey, Anal. Biochem., 117: 307-310 (1981).

Purification of Anti-CD11/CD18 Fab Antibody Fragment and Fab Variants

The anti-CD11/CD18 Fab antibody fragment and the different Fab variantswere isolated using the same extraction and purification scheme.

Extraction

Frozen cell pellets (100 g) were re-suspended at room temperature in 120mM MES buffer, pH 6.0, containing 5 mM EDTA (5 ml of buffer per g ofcell pellet) and completely disrupted by three passages through amicrofluidizer (Microfluidics Corporation, Newton, Mass.). Thehomogenate was adjusted to 0.25% (v/v) polyethyleneimine (PEI) and thesolid debris was removed by centrifugation (7280×g, 30 min, 4° C.).

ABX Chromatography

The supernatant containing the antibody fragment was diluted to aconductivity of 2.5 millisiemens with purified water, filtered through a0.22 micron filter (Suporcap-50™, Gelman Sciences, Ann Arbor, Mich.),and then loaded onto a 1.6×9.5 cm Bakerbond ABX column (J. T. Baker,Phillipsburg, N.J.) equilibrated in 50 mM MES/5 mM disodium EDTA, pH 6.0(Buffer A). The effluent was UV monitored at 280 nm. After loading, thecolumn was washed with Buffer A until the UV trace returned to baseline.Antibody fragments were eluted with a 20-column-volume gradient from 0to 100 mM ammonium sulfate in buffer A. Fractions were analyzed on acation-exchange column as described in the Analytical Methods sectionabove and pooled accordingly.

SP Sepharose High Performance (SPHP) Chromatography

The ABX pool was diluted with water for injection (WFI) to aconductivity of less than 4 mS and loaded onto a SPHP 1.6×9.2 cm column(Pharmacia-Biotech Inc., Piscataway, N.J.), equilibrated with 25 mM MOPSbuffer, pH 6.9. Separation was achieved by a 20-column-volume lineargradient from 0 to 200 mM sodium acetate in 25 mM MOPS buffer, pH 6.9.Fractions were analyzed by CSX HPLC and SDS-PAGE as described in theAnalytical Methods section above and pooled accordingly.

Formulation

The SPHP pools containing the antibody fragments were concentrated to 5mg/mL using Amicon stir cells and YM10 membrane filters (Amicon, Inc.Beverly, Mass.). The purified and concentrated antibody samples werebuffer-exchanged into phosphate buffer saline (PBS) by gel permeationchromatography on a Sephadex™ G25 (Pharmacia Biotech Inc. Piscataway,N.J.) column.

Endotoxin Determinations

Endotoxin determinations were performed with the Limulus amoebocytelysate test (Associates of Cape Cod Inc., Woods Hole, Mass.). Samplescontaining less than 2 endotoxin units (Eu) per mg of protein were usedin the pharmacokinetic studies.

Purification of the Anti-CD11/CD18 F(ab′)₂ Antibody Fragment

The F(ab′)₂ fragment was initially purified by ABX chromatography as aleucine zipper (Fab′)₂ variant [zipper-F(ab′)₂]. This construct wasengineered by adding a leucine zipper domain after the hinge region ofthe H52 heavy chain. After purification, the leucine zipper domain wascleaved by pepsin digestion after which the F(ab′)₂ was purified by SPHPand Phenyl Toyopearl™ chromatography as described below.

Extraction and ABX Chromatography of Zipper-F(ab′), Antibody Fragment

Extraction and ABX chromatography of the zipper-F(ab′)₂ antibodyfragment was carried out as described above for the Fab antibodyfragment variants.

Pepsin Digestion of Zipper-F(ab′), Antibody Fragment The ABX-purifiedZipper-F(ab′)₂ was treated with pepsin to remove the leucine zipperportion of the molecule to yield the F(ab′) antibody fragment. The ABXpurified sample was concentrated on Amicon stir cells to 5 mg/mL andthen diluted 1:3.5 with 100 mM sodium citrate buffer, pH 3.5. To thissolution, pepsin (1 mg/mL) dissolved in 100 mM sodium citrate buffer, pH3.5, was added at a pepsin-to-protein ratio of 1:12. After 4 hours atroom temperature, the mixture's pH was raised to pH 6.4 with 10% NaOH.

SPHP Chromatography of Pepsin-Treated Zipper-F(ab′), Antibody Fragment

Purification of the F(ab′)₂ antibody fragment from the leucine zipperdomain and undesired antibody fragments was accomplished by SPHPchromatography as described above for the Fab antibody fragmentvariants.

Phenyl Toyopearl™ Chromatography of SPHP-Purified F(ab′)₂ AntibodyFragment

The SPHP-purified F(ab′)₂ pool was made 1.5 M in ammonium sulfate byadding solid ammonium sulfate. The conditioned pool was then loaded ontoa Phenyl Toyopearl™ 650M (Tosohaas, Montgomeryville, Pa.) 1.6×10 cmcolumn equilibrated with 1.5 M ammonium sulfate, 50 mM sodium acetate,pH 5.4 (Buffer A). A 20-column-volume gradient was runned from 70%Buffer A to 100% 0.15 M ammonium sulfate in 50 mM sodium acetate, pH 5.4(Buffer B). The fractions were analyzed by reverse phase and CSX HPLCand SDS-PAGE as described in the Analytical Methods section above.

Formulation of F(ab′)₂ Antibody Fragment and Endotoxin Measurements

Formulation of the purified F(ab′)₂ antibody fragment was performed asdescribed above for the Fab antibody fragment variants. After endotoxindeterminations, samples containing less than 2 Eu per mg of protein wereused in the pharmacokinetic studies set forth below.

Pharmacokinetic Study of Anti-CD11/18 Constructs in Mice AfterIntravenous Administration

The objective of this single-dose pharmacokinetic study of fivehumanized huH52 anti-CD18 antibody fragments (constructs) in mice was todetermine if non-specific clearance of antibody fragments is affected byalterations to amino acids in the constant domain. Serum samples werecollected from male CD1 mice over a 24-hour period and human anti-CD18serum concentrations were measured by ELISA.

The anti-CD18 antibody fragments investigated were derived fromE.-coli-produced recombinant humanized monoclonal Fab antibody fragmentsas described above. The Fab fragment and the construct in which two Fab′subunits were joined together by two disulfide bonds were investigated.Lastly, three new versions of the original Fab were constructed byaltering amino acids in the constant domain. See the Study Design tablebelow for further description of the constructs.

The construct antigen-binding sites are directed against the CD18subunit of the CD11/CD18 glycoprotein complex on the surface ofleukocytes. These antibody fragments are chimpanzee and human-specific;therefore, the serum pharmacokinetic information obtained in miceprovides a description of the non-specific clearance of the fragments.

Because linear pharmacokinetics were expected in this study, asingle-dose level of 2 mg/kg was chosen rather than multiple-doselevels. Study Design^(a) Group Construct Number ID Construct Description1 Fab Fab fragment alone 2 Double Two Fab′ subunits joined with a doubledisulfide disulfide bond 3 Fabv1 new version 1 of the original Fabconstructed by altering amino acids in the constant domain 4 Fabv1B newversion 1B of the original Fab constructed by altering amino acids inthe constant domain 5 Fabv2 new version 2 of the original Fabconstructed by altering amino acids in the constant domain^(a)Each group consisted of 20 male mice; each mouse received a 2 mg/kgdose.

The pharmacokinetics of the five antibody constructs were studied inmale Crl:CD-1® (ICR)BR VAF/Plus® mice (approximately 20-30 g). Fivegroups, each consisting of twenty mice, received an intravenous bolusdose of 2 mg/kg via the tail vein. Blood samples were collected at 5 and36 minutes, 1, 2, 4, 8, 12, 16, 20, and 24 hours post-dose. Serum washarvested and concentrations of the antibody fragments were determinedin a MAC-1 capture ELISA as follows:

96-Well microtiter plates were coated overnight with murine anti-CD18monoclonal antibody. After overnight incubation at 4° C., plates werewashed three times with ELISA wash buffer and blocked for 1 hour withELISA diluent. ELISA wash buffer is phosphate-buffered saline(PBS)/0.05% Polysorbate™ 20. This buffer is prepared per liter as 50 mL20×PBS/1.0% Polysorbate™ 20 (a mixture obtained by dissolving 160 gNaCl, 4.0 g KCl, 22.6 g Na₂HPO₄, and 4.0 g KH₂PO₄ in glass-distilled ordeionized water, adding 10.0 mL Polysorbate™ 20 [Sigma™ P-1379 orequivalent], qs to 1000 mL, and sterile filtering using a 0.22 μm orsmaller filter), and qs to 1.0 L of distilled or deionized water, storedat ambient temperature. The expiration period is 2 weeks from the dateof preparation.

The ELISA diluent was PBS/0.5% BSA/0.05% Polysorbate™ 20/0.01%Thimerosal™/1 mM CaCl₂/1 mM MgCl₂. This diluent was prepared per literas 5.0 g bovine serum albumin (Armour™ N0068 or equivalent), 50 mL20×PBS/1.0% Polysorbate™ 20/0.2% Thimerosal™ (a mixture obtained bydissolving 160 g NaCl, 4.0 g KCl, 22.6 g Na₂HPO₄, and 4.0 g KH₂PO₄ inglass-distilled or deionized water, and adding 10.0 mL Polysorbate™ 20[Sigma P-1379 or equivalent] and 2.0 g Thimerosal™ [Sigma T-5125 orequivalent], qs to 1000 mL), 0.1% (v/v) 1 M CaCl₂ (Genentech™A3165),0.1% (v/v) 1 M MgCl₂ (Genentech™ A3167), qs to 1.0 L of distilled ordeionized water, and stored at 2-8° C., with the expiration period 1month from the date of preparation.

After blocking, the plates were washed again three times with ELISA washbuffer. Soluble MAC1 (CD11b/CD18 as described by Berman et al., J. Cell.Biochem., 52: 183-195 [1993]) was then captured out of a concentrate ofmedia, conditioned by CHO cells expressing the truncated CD11b/CD18heterodimer. After a 2-hour incubation period, the plates were washedsix times with ELISA wash buffer and 100 μL of the mouse serum samplebeing tested or the standard containing the homologous recombinant humananti-CD18 Fab were added. The mouse serum samples were first diluted1/10 in ELISA diluent and then a further ¼ into sample diluent; 100 μLwas taken from this initial 1140 dilution. Sample diluent is 10% SwissWebster Mouse serum in ELISA diluent.

Following a second 2-hour incubation, the plates were again washed sixtimes with ELISA wash buffer and 100 μL ofhorseradish-peroxidase-conjugated F(ab′)₂ directed against a human Fabwas added. After a 1-hour incubation at ambient temperature, the plateswere washed with ELISA wash buffer as described above and 100 μL ofphosphate-buffered saline, pH 7.2, containing 2.2 mmol/L orthophenylenediamine (OPD) and 0.012% (v/v) hydrogen peroxide (H₂O₂) was added toeach well. When color had fully developed, the reaction was stopped with100 μL per well of 4.5 mol/L sulfuric acid. The absorbance of the wellcontents was measured at 492 nm minus 405 nm background absorbance usingan automatic plate reader from SLT Labinstruments. Data were reduced byusing a four-parameter, curve-fitting program based on an algorithm forleast-squares estimation of non-linear parameters.

Serum concentration versus time data were analyzed utilizing anon-linear curve-fitting program and subsequent pharmacokineticsparameters were estimated. D'Argenio and Schumitzky, ADAPT II User'sGuide, Biomedical Simulations Resource, University of SouthernCalifornia, Los Angeles, Release 2, 1990.

A two-compartment model was used to characterize the serum concentrationversus time data for the five groups. See Table 2 for primary modelparameters and calculated pharmacokinetic parameters. Thetwo-compartment model fit is superimposed on the data and shown in FIGS.1A and 1B. A data listing is provided in Table 3. The volume of thecentral compartment approximated the plasma volume for all groups. TABLE2 Primary and Secondary Pharmacokinetic Model Parameter EstimatesDetermined After Administration of 2 mg/kg Constructs to Mice GroupNumber 1 2 3 4 5 Linker Fab Double S—S Fab v1 Fab v1B Fab v2 Dose 2.02.0 2.0 2.0 2.0 (mg/kg) V₁/W 44.7 53.9 51.7 42.3 49.1 (mL/kg)^(a) K_(e)(hr⁻¹)^(b) 4.22 0.486 3.35 1.89 3.86 K_(cp) (hr⁻¹)^(c) 0.431 0.581 1.214.01 1.77 K_(pc) (hr⁻¹)^(d) 1.40 1.09 1.22 3.42 1.33 CL/W (mL/ 189 26173 80 190 hr/kg)^(e) t_(1/2)α(hr)^(f) 0.14 0.37 0.14 0.08 0.11t_(1/2)β(hr) 0.57 2.5 0.84 0.92 0.83 T_(max)(min)^(g) 5.0 5.0 5.0 5.05.0 C_(max) 34 35 28 34 26 (μg/mL)^(h) C_(o)(μg/mL)^(i) 39 46 39 44 39AUC/dose/W 9.3 96 12 23 10 (hr*μg/mL)^(j) T (hr)^(k) 0.24 2.1 0.30 0.530.26^(a)Volume of the central compartment as calculated from the equation V= dose/ΣA_(i).^(b)K_(e) is the rate constant associated with the elimination ofmaterial from the central compartment.^(c)K_(cp) is the rate constant associated with the transfer of materialfrom the central to a peripheral compartment.^(d)K_(pc) is the rate constant associated with the transfer of materialfrom the peripheral to the central compartment.^(e)Weight-normalized serum clearance.^(f)t_(1/2)α and t_(1/2)β are the initial and terminal half-livesassociated with each exponential phase.^(g)Time of maximum observed concentration.^(h)Maximum observed concentrations.^(i)Zero-time concentration estimated from the disposition function asΣA_(i).^(j)Dose-normalized area under the serum concentration versus timecurve.^(k)Permanence time.

TABLE 3 Data Listing: Concentration vs. time data for 2 mg/kg humananti-CD18 constructs.^(a) Time Concentration (μg/mL) (hours) Group 1Group 2 Group 3 Group 4 Group 5 0.083 28.12 34.28 26.1 28.16 25.16 0.08333.89 34.67 28.38 33.63 26.39 0.5 4.84 26.6 4.67 10.61 4.25 0.5 5.1720.74 5.83 12.83 4.21 1 0.91 16.18 2.1 7.16 1.95 1 1.09 18.24 2.13 6.891.54 2 0.16 11.01 0.82 3.71 0.76 2 0.31 12 0.57 4.9 0.68 4 0.31 6.360.14 0.91 0.15 4  LTS^(b) 6.78 0.14 0.67 0.12 8 LTS 1.95 LTS LTS LTS 8LTS 1.66 LTS LTS LTS 12 LTS 0.71 LTS LTS LTS 12 LTS 0.88 LTS LTS LTS 16LTS 0.17 LTS LTS LTS 16 LTS 0.16 LTS LTS LTS 20 LTS 0.1 LTS LTS LTS 20LTS 0.08 LTS LTS LTS 24 LTS 0.08 LTS LTS LTS 24 LTS LTS LTS LTS LTS^(a)Concentration data represent one sample per mouse.^(b)LTS = Less than the sensitivity of the assay (0.13 μg/mL for groups1 and 3-5; 0.06 μg/mL for group 2).Results

The data are shown in FIGS. 1A and 1B, where FIG. 1A shows thepharmacokinetics of all five constructs over a time period of 0 to 5hours, and FIG. 1B shows the pharmacokinetics of all five constructsover a time period of 0 to 25 hours. The initial (or α-phase) half-livesvaried as did the terminal (β-phase) half-lives. The Fab v1B variant hada clearance of 80 mL/hr/kg, which is about three-fold higher than thatof the double-disulfide (Fab′)₂. The Fab v1, Fab, and Fab v2 hadapproximately 3-fold greater clearance over the Fab v1B and about 6-foldgreater clearance over the double-disulfide (Fab′)₂ (173, 189, and 190mL/hr/kg, respectively).

The effective molecular weight of the original Fab was 49 kD, and itsclearance was 189 mL/hr/kg.

The Fab versions 1, 1B, and 2 all have molecular weights similar to thatof the original Fab, yet version 1B was cleared from the serum 2-foldmore slowly. Thus, alterations of the amino acid sequence in the Fabconstant domain affect clearance. The effect seen on beta-phasehalf-life shows that with the two least-successful variants 1 and 2,there was a detectable effect that was not sufficient to increasesignificantly overall permanence time.

1. Nucleic acid encoding a polypeptide variant of a polypeptide ofinterest which polypeptide of interest is cleared from the kidney anddoes not contain a Fc region of an IgG, which variant comprises asalvage receptor binding epitope of an Fc region of an IgG, and whichvariant has a longer in vivo half-life than the polypeptide of interest.2. The nucleic acid of claim 1 wherein the polypeptide of interestcontains an Ig domain or Ig-like domain that is not a CH2 domain.
 3. Thenucleic acid of claim 2 wherein the epitope is contained within the Igdomain or Ig-like domain.
 4. The nucleic acid of claim 3 wherein the Igdomain or Ig-like domain comprises a CH1 domain.
 5. The nucleic acid ofclaim 3 wherein the epitope is taken from one or two loops of the Fcregion and transferred to the Ig domain or Ig-like domain.
 6. Thenucleic acid of claim 5 wherein the epitope is taken from the CH2 domainof the Fc region and transferred to the CH1, CH3, or V_(H) region, ormore than one such region, of an Ig or to a Ig-like domain.
 7. Thenucleic acid of claim 5 wherein the epitope is taken from the CH2 domainof the Fc region and transferred to the C_(L) region or V_(L) region, orboth, of an Ig or to an Ig-like domain.
 8. The nucleic acid of claim 3wherein the polypeptide of interest is a Fab, a (Fab′)₂, a diabody, a Fvfragment, a single-chain Fv fragment, or a receptor.
 9. The nucleic acidof claim 8 wherein the polypeptide of interest is an LFA-1 antagonist.10. The nucleic acid of claim 9 wherein the polypeptide of interest is aFab or (Fab′)₂ of an anti-LFA-1 antibody.
 11. The nucleic acid of claim10 wherein the polypeptide of interest is an anti-CD18 Fab or anti-CD18(Fab′)₂.
 12. The nucleic acid of claim 11 wherein the polypeptide ofinterest is human or humanized.
 13. The nucleic acid of claim 1 whereinthe epitope comprises the sequences: HQNLSDGK (SEQ ID NO: 1), HQNISDGK(SEQ ID NO: 2), HQSLGTQ (SEQ ID NO: 11), or VISSHLGQ (SEQ ID NO: 31) andPKNSSMISNTP (SEQ ID NO: 3).
 14. The nucleic acid of claim 13 wherein theepitope is fused to the polypeptide of interest.
 15. The nucleic acid ofclaim 14 wherein the polypeptide of interest is growth hormone or nervegrowth factor.
 16. A replicable vector comprising the nucleic acid ofclaim
 1. 17. A host cell comprising the nucleic acid of claim
 1. 18. Ahost cell that is transformed with the nucleic acid of claim
 1. 19. Amethod for producing a polypeptide variant comprising culturing the hostcells of claim 1 in a culture medium and recovering the variant from thehost cell culture.
 20. The method of claim 19 wherein the variant isrecovered from the host cell culture medium.